Title of Invention

"AN IMPROVED PROCESS FOR THE PREPARATION OF CAROTENOIDS FROM ENCYSTED HAEMATOCOCCUS CELLS"

Abstract

An improved process for the preparation of carotenoids from Heamatococcus cells characterized in simple method of extraction of said caratonoids by using acidified aqueous solution, which comprises suspending encysted Heamatococcus cells in acidified aqueous solution having concentration in the range of 0.01 N to N at a temperature range of 8-90° C for a period of 30 seconds to 15 minutes, separating the encysted cells by conventional centrifugation and getting the desired carotenoid preferably astaxanthin by solvent extraction of said acidified aqueous solution obtained after centrifugation.

Full Text

The present invention relates to an improved process for the preparation of carotenoids from encysted Haematococcus cells, Astaxanthin is a ketocarotenoid (3,3'-dihydroxy p ,p carotene- 4,4' dione) used extensively in aquaculture and poultry as pigmentation source. The carotenoid imparts distinctive orange-red coloration to the animals especially in salmon, trout and egg yolk and contributes to consumer appeal in the market place thereby adds value to the products. Carotenoids also have important metabolic functions in animals and man including conversion to vitamin A, enhancement of the immune response and protection against diseases such as cancer by scavenging of oxygen radicals (Johnson, An 1991, Astaxanthin from microbial sources, Critical Reviews in Biotechnology, 11: 249-326). Since animals can not synthesize carotenoids, the pigments must be supplemented in the feeds of farmed species. The growing trend towards using natural sources of feed nutrients, the health benefits of carotenoids placed greater demand for natural sources of carotenoids. Although astaxanthin has been identified in several microorganisms, the primary biological sources of astaxanthin currently being considered for industrial production are the green alga Haematococcus pluviads and related sps, the heterobasldiomyces yeast Phaffia rfxidozyma and crustacean extracts. With the large quantities of crustacean waste generated each year, much effort has gone into using this as a carotenoid source in fish aquaculture. The major problem in using this waste is the high level of ash and the low energy content of the shells (Storebakken T (1988) Aquaculture 70: 193). The basidiomycetes yeast Phaffia contains 0.02 to 0.03% astaxanthin on dry weight basis and considerable progress was made to increase the astaxanthin content by using mutants raised with chemical mutagens. However instability of mutants and nonreproducible production levels in reactors were the major limitations. Haematococcus, the green alga is another potential organism for astaxanthin production. It is a unicellular, biflagellate, motile organism which can grow both autotrophically and heterotrophicaliy. It forms a cyst under unfavourable growth conditions. This encystment is accompanied by the synthesis and accumulation of carotenoids especially astaxanthin. During the encystment process, the relative quantities of individual carotenoids change considerably from 75-80% lutein and 10-20% carotene to over 80-90% astaxanthin in encysted cells. Although the astaxanthin content n Haematococcus accounts to more than 2% on dry weight basis, the tough cell wail of cyst cells hinders solvent extraction and intestinal absorption of the pigment from the intact algal biomass. Methods of cell wall disruption which have been applied include mechanical breakage, chemical hydrolysis and lytic enzymes ( Okabue, RN and -ewis MJ 1983 Biotechnol.Lett 5: 731-736., Grung M, D'Souza, FML, Borowitzka M, and Jaaen Jensen S 1992, J appi Phycol 4: 165-171) either denature astaxanthin or are cumbersome and difficult to apply on a large scale. Therefore the present invention provides an alternate improved process for the extraction of carotenoids from encysted Haematococcus cells which facilitated extraction of carotenoids with out homogenisation of cells or use of lytic enzymes. Procedures currently reported for the extraction of astaxanthin from microorganisms are the following Reference may be made to the procedures of Davies 1976 (carotenoids. In Goodwin T.W (Ed) Chemistry and biochemistry of plant pigments. Academic press, London, PP. 38-166) and Lichtenthaler 1987 (Chlorophylls and Carotenoids : pigments of photosynthetic biomembranes. Methods of Enzymology 148: 350-382) wherein the extraction of carotenoids included sonication/homogenisation of cells in acetone. The drawback is that these procedures limits to routine estimation of carotenoids and the carotenoid extract contains chlorophyll. Reference may be made to the procedure of S.Boussiba and A.Vonshaik (Astaxanthin accumulation in the green alga Haematococcus pluviaiis. Plant Cell Physiol 32:1077-1082 1991) wherein the encysted cells were first treated with a solution of 5% KOH in 30% methanol to destroy the chlorophyll. The remaining pellet was repeatedly extracted with DMSO (dimethyl sulfoxide) to recover astaxanthin. The drawback is that the extraction of astaxanthin with DMSO requires repeated extractions and it is a slow process for complete extraction. The astaxanthin extracted using DMSO can not be used directly and requires extraction with solvent such as ethyl acetate and hexane and this procedure limits to estimation and analysis purposes. Reference may be made to the method of M Kobayashi et al 1997 (M. Kobayashi, Y.Kurimura, Y. Sakamoto and Y.Tsuji Selective extraction of astaxanthin and chlorophyll from the green alga Haematococcus pluviaiis. Biotechnology Techniques 11: 657-660) wherein the encysted cells are heat treated with acetone : water (0-100%) at 80 °C for 2 minutes and subsequently subjected to lyophilisation or treatment with lytic enzymes and found an increase in extractability of carotenoids. Heat treatment of encysted cells with acetone water (0-100%) alone did not facilitated carotenoid extraction and only after subsequent treatments such as lyophilisation or treatment with lytic enzymes such as Kitalase (p-1,3 giucanase from Rhizoctonia solani), Cellulase onozuka -RS (p-1,4 giucanase from Tiichoderma sp) and abalone acetone powder (p-glucuronidase) enhanced extractability of astaxanthin. Although astaxanthin extraction was achieved with out sonication of cells, treatment of lyophilisation or lytic enzymes adds to the cost and difficult to apply for large scale applications and complete extraction was not achieved by this method. Reference may be made to US patent (US 1989 4871551) wherein encysted Haematococcus cells were ground at cryogenic temperatures typically by combining the cells with liquid nitrogen in a suitable impact mill to particle size below about 5.5 microns, the drawback is that grinding of cells at cryogenic temperatures is expensive and the carotenoids in powder are liable for degradation due to oxidation. To prevent this antioxidant compounds have to be incoporated throughout the process. Reference may be made to US patent (US 1993 5179012) wherein the microaigae selected from rhodophyceae and chiorophyceae after separating the microaigae from culture medium and dispersing in a solution and crushing the dispersed microorganisms and adding solvent to the solution to solubilise the antioxidants produced by the microorganisms. The drawback is that crushing of cells liberates many hydrophilic and hydrophobic components of cells and while extracting for carotenoids some of the hydrophobic components may also be extracted along with carotenoids and may add another step for purification of carotenoids. Main object. of the present invention is to provide an improved process for the preparation of carotenoids from encysted Haematococcus cells which obviates the drawbacks as detailed above. Accordingly the present invention provides an improved process for the preparation of carotenoids from Heamatococcus cells characterized in simple method of extraction of said caratonoids by using acidified aqueous solution , which comprises suspending encysted Heamatococcus cells in acidified aqueous solution having concentration in the range of 0.01 N to 10 N at a temperature range of 8-90 ° C for a period of 30 seconds to 15 minutes , separating the encysted cells by conventional centrifugation and getting the desired carotenoid preferably astaxanthin by solvent extraction of said acidified aqueous solution obtained after centriftjgation. In an embodiment of the present invention the acidified aqueous solution may be selected from mineral acids and organic acids such as phosphoric acids , hydrochloric acid and sulphuric acid , citric acid , acetic acid , formic acid , oxalic acid , tartaric acid. In another embodiment of the present invention the solvent used for the extraction of carotenoids may be selected from acetone , ethyl acetate , hexane , diethyl ether and chloroform. The novelty of the invention is that treatment of encysted Haematococcus cells in acidified aqueous solution facilitated the extraction of carotenoids/astaxanthin without homogenization or grinding of the encysted cells which possibly could be due to break up of crucial linkages among the components of the cyst which are otherwise resistant to alkali, pressure treatment, detergents such as Tween -20 , triton X-100 , C-TAB and to most of the lytic enzymes . Another advantage is that the encysted cells can be preserved after treatment till their further use. This treatment avoids complete breakage of cells and the carotenoids could easily be extracted by the solvent as and when required for use. The microscopic observation of the cells thus treated and extracted showed carotenoids extracted with solvent from cells was found to be unstable due to oxidation and difficult to store while pretreated cells in the present process can be stored for longer period without loss of pigments . The following examples are given by way of illustration of the present invention and therefore should not be construed to limit the scope of the present invention. EXAMPLE -1 1 gram of encysted Haematococcus cells were suspended in 100 ml of acidified aqueous solution of citric acid at a concentration of 6N at 45°C for 10 minutes duration and the cells were separated at 8000 rpm for 7 minutes and the supernatant discarded. The treated cells were extracted with acetone and the concentration of carotenoid, astaxanthin and chlorophyll were determined according to the method of Lichtenthaler 1987 (Chlorophylls and carotenois: Pigments of photosynthetic biomembranes. Methods in Enzymoiogy 148: 350-382) by reading absorbance at 450, 470, 480, 650 and 661.5nm using Shimadzu spectrophotometre. The extractability of astaxanthin was calculated using the formula; Extractability {%) = free astaxanthin (µg m-1)/total astaxanthin (µg m-1) X 100. Free astaxanthin was determined as the amount extractable by direct 90% acetone treatment of cells for 1 hour while total astaxanthin was determined as the amount extractable by 90% acetone for 1 hour after homogenisation of the cells (Kobayashi et al 1997, M. Kobayashi, Y.Kurimura, Y.Sakamoto and Y.Tsuji Selective extraction of astaxanthin and chlorophyll from the green alga Haematococcus pluviaiis, biotechnology Techniques 11: 657-660). The extractability of astaxanthin was found to be 3.8%. EXAMPLE - 2 500 milligrams of encysted Haematococcus cells were suspended In 50 m! of acidified aqueous solution of acetic acid at a concentration of 2N at 70°C for 12 duration and the cells were separated at 6000 rpm for 8 minutes and the supernatant discarded. The treated cells were extracted with acetone and the concentration of carotenoid, astaxanthin and chlorophyll were determined according to the method of Lichtenthaler 1987 (Chlorophylls and carotenois: Pigments of photosynthetic biomembranes. Methods in Enzymology 148: 350-382) by reading absorbance at 450, 470, 480, 650 and 661.5nm using Shimadzu spectrophotometre. The extractability of astaxanthin was calculated using the formula: Extractability (%) = free astaxanthin ((og ml-1) / total astaxanthin (µg ml-1) X 100. Free astaxanthin was determined as the amount extractable by direct 90% acetone treatment of cells for 1 hour while total astaxanthin was determined as the amount extractable by 90% acetone for 1 hour after homogenisation of the cells (Kobayashi et al 1997, M. Kobayashi, Y.Kurimura, Y. Sakamoto and Y.Tsuji. Selective extraction of astaxanthin and chlorophyll from the green alga Haematococcus pluvialis, Biotechnology Techniques 11: 657-660). The extractability of astaxanthin was found to be 20%. EXAMPLE -3 500 milligrams of encysted Haematococcus cells were suspended In 70 ml of acidified aqueous solution of hydrochloric acid at a concentration of 0.5N at 50°C for 15 minutes duration and the cells were separated at 3000 rpm for 10 minutes and the supernatant discarded. The treated cells were extracted with diethyl ether and the concentration of carotenoid, astaxanthin and chlorophyll were determined according to the method of Lichtenthaler 1987 ( Chlorophylls and carotenois: Pigments of photosynthetic biomembranes. Methods in Enzymology 148: 350-382) by reading absorbance at 450, 470, 480, 650 and 661.5nm using Shimadzu spectrophotometre. The extractabiiity of astaxanthin was calculated using the formula: Extractability (%) = free astaxanthin (µg ml-1) / total astaxanthin (µg ml-1) X 100. Free astaxanthin was determined as the amount extractable by direct 90% acetone treatment of cells for 1 hour while total astaxanthin was determined as the amount extractable by 90% acetone for 1 hour after homogenisatlon of the cells (Kobayashi et al 1997, M. Kobayashi, Y.Kurimura, Y. Sakamoto and Y.Tsuji. Selective extraction of astaxanthin and chlorophyll from the green alga Haematococcus p/uviafis, Biotechnology Techniques 11: 657-660). The extractability of astaxanthin was found to be 80%. EXAMPLE -4 800 milligrams of encysted Haematococcus cells were suspended in 75 ml of acidified aqueous solution of formic acid at a concentration of 4N at 60°C for 10 minutes duration and the cells were separated at 5000 rpm for 9 minutes and the supernatant discarded. The treated cells were extracted with acetone and the concentration of carotenoid, astaxanthin and chlorophyll were determined according to the method of Lichtenthaler 1987 (Chlorophylls and carotenois: Pigments of photosynthetic biomembranes. Methods in Enzymology 148: 350-382) by reading absorbance at 450, 470, 480, 650 and 661.5nm using Shimadzu spectrophotometre. The extractability of astaxanthin was calculated using the formula: Extractability (%) = free astaxanthin (µg m-1) / total astaxanthin (µg m-1) X 100. Free astaxanthin was determined as the amount extractable by direct 90% acetone treatment of cells for 1 hour while total astaxanthin was determined as the amount extractable by 90% acetone for 1 hour after homogenisation of the cells (Kobayashi ef al 1997, M. Kobayashi, Y.Kurimura, Y. Sakamoto and Y.Tsuji. Selective extraction of astaxanthin and chlorophyll from the green alga Haematococcus pluvia/is, Biotechnology Techniques 11: 657-660). The extractability of astaxanthin was found to be 15%. EXAMPLE -5 600 milligrams of encysted Haematococcus cells were suspended in 60 mi of acidified aqueous solution of hydrochloric acid at a concentration of 2N at 70°C for 10 minutes duration and the cells were separated at 7000 rpm for 8 minutes and the supernatant discarded. The treated ceils were extracted with diethyl ether and the concentration of carotenoid, astaxanthin and chlorophyll were determined according to the method of Lichtenthaler 1987 (Chlorophylls and carotenois: Pigments of photosynthetic biomembranes. Methods in Enzymology 148: 350-382) by reading absorbance at 450, 470, 480, 650 and 661.5nm using Shimadzu spectrophotometre. The extractability of astaxanthin was calculated using the formula: Extractability (%) = free astaxanthin (µg m-1)/total astaxanthin (µg m-1) X 100. Free astaxanthin was determined as the amount extractable by direct 90% acetone treatment of cells for 1 hour while total astaxanthin was determined as the amount extractable by 90% acetone for 1 hour after homogenisation of the cells (Kobayashi et ai 1997, M. Kobayashi, Y.Kurimura, Y. Sakamoto and Y.Tsuji. Selective extraction of astaxanthin and chlorophyll from the green alga Haematococcus pluvialis, Biotechnology Techniques 11: 657-660). The extractability of astaxanthin was found to be 95%. EXAMPLE-6 500 milligrams of encysted Haematococcus cells were suspended in 50 ml of acidified aqueous solution of tartaric acid at a concentration of 5N at 80°C for 12 minutes duration and the ceils were separated at 6000 rpm for 8 minutes and the supernatant discarded. The treated cells were extracted with hexane and the concentration of carotenoid, astaxanthin and chlorophyll were determined according to the method of Lichtenthaler 1987 (Chlorophylls and carotenois: Pigments of photosynthetic biomembranes Methods in Enzymoiogy 148: 350-382) by reading absorbance at 450, 470, 480, 650 and 661.5nm using Shimadzu spectrophotometre. The extracteibility of astaxanthin was calculated using the formula: Extractability (%) = free astaxanthin (µg m-1) / total astaxanthin (µg m-1) X 100. Pree astaxanthin was determined as the amount extractable by direct 90% acetone treatment of cells for 1 hour while total astaxanthin was determined as the amount extractable by 90% acetone for 1 hour after homogenisation of the cells (Kobayashi et al 1997, M. Kobayashi, Y.Kurimura, Y. Sakamoto and Y.Tsuji. Selective extraction of astaxanthin and chlorophyll from the green alga Haematococcus pluvialis, Biotechnology Techniques 11: 657-660). The extractability of astaxanthin was found to be 28%. The main advantages of the present invention are 1 Complete extraction of carotenoid / astaxanthin from encysted Haematococcus cells was achieved without homogenisation or grinding of the cells. 2 The process eliminated the use of lytic enzymes which is otherwise expensive and cumbersome to scaleup. 3 The process developed for carotenoid extraction easy to scaleup. 4 The tough ceil wall of cyst cells hindered solvent extraction with out homogenisation and the cysts are found to be resistant to aikali treatment, pressure treatment and with detergents. 5 The process developed facilitated solvent extraction of carotenoids with out further sonication or lytic enzyme treatments. 6. The treated encysted cells can be preserved as such till their further use and also the treated cells can be incorporated in the feeds directly without extraction of the carotenoids with solvent. 7 After extraction of carotenoids / astaxanthin / the cells can be used as a by-product for any feed purposes. 8 The carotenoid / astaxanthin extract contained traces or nil chlorophyll. We Claim: 1. An improved process for the preparation of carotenoids from Heamatococcus cells characterized in simple method of extraction of said caratonoids by using acidified aqueous solution , which comprises suspending encysted Heamatococcus cells in acidified aqueous solution having concentration in the range of 0.01 N to 10 N at a temperature range of 8-90 ° C for a period of 30 seconds to 15 minutes , separating the encysted cells by conventional centrifugation and getting the desired carotenoid preferably astaxanthin by solvent extraction of said acidified aqueous solution obtained after centrifugation. 2. An improved process as claimed in claim -1 wherein acid used for getting the aqueous solution of acids is selected from mineral acids such as phosphoric acid , hydrochloric acid and organic acids such as acetic acid , formic acid , oxalic acid, tartaric acid. 3. An improved process as claimed in claims 1 & 2 wherein the solvent used for extraction of carotenoid is selected from acetone , ethyl acetate , hexane , diethyl ether, chloroform. 4. An improved process for the preparation of carotenoids from encysted Haematococcus cells substantially as herein described with references to the examples. We Claim: 1. An improved process for the preparation of carotenoids from Heamatococcus cells characterized in simple method of extraction of said caratonoids by using acidified aqueous solution , which comprises suspending encysted Heamatococcus cells in acidified aqueous solution having concentration in the range of 0.01 N to 10 N at a temperature range of 8-90 ° C for a period of 30 seconds to 15 minutes , separating the encysted cells by conventional centrifugation and getting the desired carotenoid preferably astaxanthin by solvent extraction of said acidified aqueous solution obtained after centrifugation. 2. An improved process as claimed in claim -1 wherein acid used for getting the aqueous solution of acids is selected from mineral acids such as phosphoric acid , hydrochloric acid and organic acids such as acetic acid , formic acid , oxalic acid, tartaric acid. 3. An improved process as claimed in claims 1 & 2 wherein the solvent used for extraction of carotenoid is selected from acetone , ethyl acetate , hexane , diethyl ether, chloroform. 4. An improved process for the preparation of carotenoids from encysted Haematococcus cells substantially as herein described with references to the examples.

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