Title of Invention

A METHOD OF THE EXTRACTION AND PURIFICATION OF NATURAL MIXED CAROTENOID FROM MICROALGAE

Abstract

The process of the present invention relates to the extraction and purification of both a natural mixed carotenoid product in oil form and an crystalline form wherein the crystalline form contains predominantly the alltrans isomer of betacarotene and the other minor carotenoids are alpha carotene, lutein, zeaxanthin and the cis isomer of betacarotene and the oil form contains predominantly the cis isomer of betacarotene, with substantial quantities of lutein and zeaxanthin along with alphacarotene from solid wet biomass of algae containing about 80% moisture, preferably from algae of the genus Dunaliella. More particularly, the present invention relates to a single solvent process whereby both natural and colorant products lie along the same process line. The nutritional product contains the natural array of carotenes and xanthophylls found in both the oil form ans the crystalline form.

Full Text

The present invention relates to a method of the extraction and purification of natural mixed carotenoid from miefoalgae. An object of the present invention is extraction and purification done by using a single solvent and a single step is included to obtain both forms of mixed carotenoids. PRIOR ART: Carotenoids are the most widespread class of naturally occurring pigments in nature, present without exception in photosynthetic tissue and ■ t occurring with no definite pattem in non-photosynthetic tissues such as root, flower petals, seeds and fruits. They are also found in algae, fungi, yeasts, molds, mushrooms and bacteria, and in many cases they .are the major pigment in the exoskeleton of aquatic and avian species. Carotenoids and/or carotenes derive their names from the fact that they constitute the major pigment in the carrot root, one of the first foods observed to possess this class of pigments. carotenoid pigment is a source of vitamin A, provides nutritional supplements to man. Vitamin A or retinol has long been known to be necessary to the biochemistry of vision and to the proper ftinction of the epithelial tissues. Deficiencies of vitamin A (nay lead to reduced visual sensitivity, such as, night blindness and in extreme cases complete blindness or reduced resistance to infection through epithelial surfaces. Beta-carotene is a surrogate for vitamin A. According to reports and nical studies, Beta-carotene has an important chemopreventive or chemo-stponing agent in aging, immune deficiency, senile cataracts and in several ler types of cancer. A variety of different procedures for isolating and purifying Beta-otenes from plant materials are in used. In the case of extracting Beta-otene from palm oil, the known methods can be classified as follows: Extraction Method: Patent Application No: GB 657,682; U.S. Pat. No, 2,460,796; U.S. Pat. No. 2,440,029; US 2,572,467; and U.S. Pat. No. 2,652,433. are claiming the extraction of carotene by saponification, wherein the palm oil is saponified to give soap, glycerol and a nonsaponifiable fraction containing carotenes. Iodine method, wherein iodine is added to a solution of palm oil in petroleum ether, an insoluble precipitate of carotene di-iodides is formed. The iode compounds when treated with sodium thiosulfate however yield iso-carotenes or dehydrocarotenes which are not natural. Urea process, wherein triglycerides are broken down to fatty acids and methyl esters which then fomi insoluble compounds with urea thiourea, leaving the carotenoids in the remaining liquid. Extraction using Fuller's earth or activated carbon, wherein recovery of the carotenoids from the earth gives oxidized or isomerized carotenoids. For examples of such, see Patent Application Nos.: GB 691,924; GB 1,563,794; and U.S. Pat. No. 2,484,040. Extraction by selective solvents has been carried out using propane or furfural, see U.S. Pat. No. 2,432,021. Molecular distillation at 10-3 to 10-4 mm Hg. A process of trans esterification followed by molecular distillation of the ester. Fractions collected at 230.degree. C. have a carotene content of about five times that of the original oil. Liaaen-Jensen, S., The Carotenoids (O. Isler, ed), Birkhauser Verlag, Basel, p. 61 (1971), and Britton, G., Methods Enzymol., 111:113 (1985) described the extraction of carotenoids from plant and animal tissues. In brief, oxygen, light and heat are the most destructive factors and should be carefiiUy avoided. The presence of oxygen during extraction may result in the formation of oxidative artifacts, or the disappearance of compounds, such as, phytofluene, due to complete oxidative breakdown. Furthermore, light and heat may cause isomerization. Peroxide-free solvents and an antioxidant such as butylated hydroxytoluene (BHT) should always be used during the extraction of carotenoids. If possible, exposure to acid and alkali (except for saponification) should also be avoided. U.S. Pat. No. 4,680,314 to Nomura et al., discloses a process for concentrating algae and extracting Beta-carotene with an edible oil such as vegetable oil at elevated temperatures, that is, 66 degree to 100 degree C. Carotene concentration in the oil extract is reported to be on the order of 1.9%. U.S. Pat. No. 4,439,629 to Ruegg et al., discloses a process for treating algae with calcium hydroxide at an elevated temperature to saponify the chlorophyll and produce a residue which is then filtered, dried, and extracted with a solvent, such as a halogenated hydrocarbon or an aliphatic or aromatic hydrocarbon, and recrystallized to yield enriched all-tram Beta-carotene. US Pat Application no 20020082459 of Bailey, David T; et al. discloses a process to get high purity betacarotene as the all trans form and a natural mixed carotenoid oil form from the algae of the genus Dunaliella. More particularly, the present invention relates to a single solvent process whereby both natural and colorant products lie along the same process line. The algae is first microfiltered and then the emulsifying agents namely the glycerol is separated out and then is subsequently extracted using the single solvent system to isolate and purify carotenoids. Presently the methods used whereby Beta-carotenes are extracted and isolated from various plant materials. However, each process disclosed involves multiple steps using various solvents or with a single solvent reused in multiple steps. Consequently, the disclosed processes are not easily scaled up to an efficient commercial process where disposal considerations of various solvents play an important role in the overall feasibility of the process where multiple solvents are used and in cases where a single solvent used a number of steps are performed back and forth to achieve a final product which is energy intensive and time consuming. There is still a need, therefore, for a process and procedure for isolating and purifying natural carotenoids as a mixed carotenoid in the crj^stalline form or the oil form. Field of Invention: The present invention relates to an improved method of extraction and purification of natural mixed carotenoid product in crystalline form and an oily form, of which, the crystalline form containing predominantly the all tram form along with other carotenoids and the oily form predominantly containing the cis form along with the other carotenoids in substantial quantit>^ It is an object of the present invention to provide a simplified method for the extraction , isolation and purification of mixed carotenoid compounds. It is a further object of the present invention to provide a single solvent and a single step extraction process whereby both crystalline and oil forms of mixed carotenoids are obtained. It is also an object of the present invention is to use the extraction step as the first and final step for the use of extraction of mixed carotenoid forms of oil and crystal. It is also an object of the of the present invention is the use of the same solvent finally to wash the mixed carotenoid crystal to a purity of all trans form >85% pure. DETAILED DESCRIPTION OF THE INVENTION: The present invention relates to a single step solvent process whereby both crystalline and oil forms of natural mixed carotenoid in the same process line. The process includes contacting a plant material with alum or ferric chloride so that the size of the algae is handlable. The aggregation of the algal cells called floes are large in size and are easily centrifuged. Thus centrifuged algae is directly made to come in cantact with the solvent, and the resulting extracted solvent is removed in vacuo obtaining a extract containing a mixture of carotenoids . The concentrated extract is filtered to obtain the crystal form of mixed carotenoid containing predominantly all trans Beta-carotene and the filtrate containing predominantly the cis form of Beta-carotene. The crystalline mixed carotenoid extract is washed with ethyl acetate at room temperature and dried in vacuo to obtain a mixed carotenoid crystal containing all trans fomi of Beta-carotene >85%, then extract is filtered to remove suspended fine plant materials and then heated to evaporate substantially all of the solvent resulting in an oil. The oil may be used as a nutritional product or as a precursor to a colorant product. If a colorant product is desired, the oil is ftirther heated, thereby isomerizing the cis Beta-carotene compounds to all trans Beta-carotene isomers. The all trans Beta-carotene compounds are then crystallized with the addition of cold solvent. The Beta-carotene containing compositions of the present invention is prepared from a variety of plant materials, such as algae, vegetables such as spinach, broccoli, alfalfa, and other plants. Preferably the plants are algae. Among the algae, the preferred classes are Chlorophyta (green algae), of which the preferred genus is Dunaliella. Other genera are also used so long as carotene can be produced in relatively large quantities. Cultivation techniques may significantly increase the amount of carotene present in each algal cell or body. In the preferred embodiment, the slurry is dewatered and concentrated by first flocculation increasing the floe size to envisage a basket centrifuge to dewater the algae. Following this concentration step of the alga is extracted using a single solvent as a initial and final step. The extraction and subsequent purification procedures are typically performed under low light intensity and under vacuum or an atmosphere of inert ga*? (e.g., nitrogen) to maximize recovery of non-oxidized carotenes. The extraction solvent used in the present invention is ethyl acetate. In the extraction step, the temperature of extraction is at 35 - 40 Deg C. Carotenoid extraction is carried out in a blender preferably a dry blender type with an rpm of 75 - 150 preferably 100 rpm for a period of 30 min to 45 min. The solvent after extraction is immediately transferred to another container. There is no phase separation, as the extraction is performed on the wet semisolid biomass. Three extractions are performed like wise for a period of 30 to 45min and the extracts are pooled in the same container. The pooled organic extract is then filtered to remove any algal material and then concentrated in vacuo to complete dryness leaving behind an oil which is the concentrated mixed carotenoid extract. This extract is filtered under vacuum separating the crystalline form of natural mixed carotenoid containing predominantly the all trans form of betacarotene and the filterate the oil form of natural mixed carotenoid containing predominantly the cis isomer of betacarotene. EXAMPLE 1 Extraction of the forms of Natural Mixed carotenioid forms. To 300fCgs of wet semisolidfbiomass having a moisture content of about 70 - 80% is contacted with 175 litres of ethyl acetate. The extraction is performed in a blender for a period of 30 min and the solvent is transferred to a container. The extraction is performed twice with the same quantity of solvent and the solvent is pooled to the same container. The pooled extract is then filtered to remove algal mass and then/(5oncentrated. The concentration is performed at 40 - 50 Deb c umi^ reduced pressure to attain a final mixed carotenoid concentrate of 2354gms. Thus obtained extract is allowed to stand for 4 hours and then fifterted to obtain 836gms of the crystalline form of natural mixed carotenoid. The fijt^te is the oil of 15K) gms of natural mixed carotenoid. 2ne crystalline mixed carotenoid form is u« of ethy|/acetat€i at/room temperature ujraer vacuum and dried in^^a^uum tor 24 Tl{e/composition CH the crystalline natural mixed carotenoid form are predominantly alltram betacarotene (85% pure) with the 5 - 6 % of alpha carotene and ds betacarotene with 1 - 2 % of lutein and zeaxanthin. The composition of the oil natural mixed carotenoid are predominantly the cis betacarotene isomer (60-70 %) with all trans isomer of 20 - 25%, with lutein and zeaxanthin contributing 5 - 6%, We claim, 1. A method of the extraction and purification of natural mixed carotenoid from microalgae comprising following steps: (a) contacting an algae with alum or ferric chloride; (b)centrifugating algal floes; (c) contacting wet algae biomass having 70- 80% moisture for a selected period of time with the said solyent, and solubilizing .- * carotenoids (d) collecting and filtering said crude extract; (e) evaporating said solvent from crude extract and forming an oil containing carotenoids; » (f) separating filtrate and crystalline form by filtering mixed crystalline form obtained in step (e); (g) washing the crystalline form using the same solvent. 2. A method of the extraction and purification of natural mixed carotenoid from microalgae as claimed in claim 1 wherein said solvent is ethyl acetate. 3. A method of the extraction and purification of natural mixed carotenoid from microalgaeas claimed in claim 1 wherein said algae is Dunaliella salina. A method of the extraction and purification of natural mixed carotenoid from microalgae as claimed in claim 3 wherein said alga is a whole alga. 4 A method of the extraction and purification of natural mixed carotenoid from microalgae as claimed in claim 1 wherein said selected period of time is from about 1 hour to 3 hours. A method of the extraction and purification of natural mixed carotenoid from microalgae as claimed in claim 1 wherein the said crystalline form is a mixed carotenoid containing predominantly the all trans isomer of betacarotene and the other minor carotenoids are alpha carotene, lutein, zeaxanthin and the cis isomer of betacaroten. A method of the extraction and purification of natural mixed carotenoid from microalgae as claimed in claim 1 wherein the said oil filtrate is a mixed carotenoid containing predominantly the cis isomer of betacarotene, with substantial quantities of lutein and zeaxanthin along with alpha carotene. A method of ttie extraction and purification of natural mixed carotenoid from microalgae as claimed in claim Ithe said concentration is performed under reduced pressure at 40 to 50 °C. A method of the extraction and purification of natural mixed carotenoid from microalgae as claimed in claim 1 wherein the washing of the crystalline form is performed at room temperature. 10. A method of the extraction and purification of natural mixed carotenoid from microalgae as claimed is herein substantially described herewith forgoing description smd example.

Documents:

393-che-2003-abstract.pdf

393-che-2003-claims.pdf

393-che-2003-correspondnece-others.pdf

393-che-2003-correspondnece-po.pdf

393-che-2003-description(complete).pdf

393-che-2003-description(provisional).pdf

393-che-2003-form 1.pdf

393-che-2003-form 26.pdf

393-che-2003-form 3.pdf

393-che-2003-form 5.pdf

393-che-2003-form 8.pdf