Title of Invention

"AN IMPROVED PROCESS FOR EXTRACTION OF CAROTENOIDS"

Abstract

The present invention relates to an effective method of retention of carotenoids in Rhodotorula grctcilis cells and its extraction. There is a considerable interest worldwide in the development of food colourants from natural sources. This is apparently due to strong consumer demand for more natural colourants is associated with their image of being healthy and of good quality. In addition there are a lot of concerns about safety of certain additives and legislation governing the use of synthetic colours.

Full Text

The present invention relates to an improved process for extraction of carotenoides from Rhodotorula gracilis cells. There is a considerable interest worldwide in the development of food colourants from natural sources. This is apparently due to strong consumer demand for more natural Colourants is associated with their image of being healthy and good quality. In addition there are a lot of concerns about safety of certain additives and legislation governing the use of synthetic colours. As a result of deficiencies of natural food colourants such as instability against light, heat, pH, the demand for alternative sources of natural pigment is repeatedly raised by the food industry Hence the search for alternative colourants- the microbial sources. During the past ten years,much interest and effort has been devoted to designating and developing microbial source for industrially important carotenoides. Microbial synthesis could provide a significant proportion of the pigment used in terrestrial agriculture and marine agriculture and potentially as human nutrient or neutraceuticals. Caroicnoids arc group of pigments that contribute to the yellow, orange or red colouration of fruits and vegetables. They are telra terpcnoids compounds which contain 40 Carbon atoms arranged in a repeating pattern of 5 carbon units. As pigments in food products, fi- carotene and other carotenoids are very useful, because of their high colour intensity and low toxicity. Natural colours such as carotenoids have various biological functions apart from acting as a colourant. For many years, the health benefits of carotenoids focused predominantly on the vitamin A activity of P-carotene. Today, many natural carotenoids are being investigated for their role in a \anety of health benefits. Cumulative evidences from epidemiological studies shows a lower risk to a variety of chronic diseases including various cancer, cardiovascular disease, age related macular degeneration and cataracts. Most of these carotenoids find their application in food, feed, nutritional supplement, cosmetics and pharmaceutical industries. In, food, they arc widely used in beverages, dry canned soups, dairy products such as cheese, ice cream and yogurt, desserts like jellies, pudding, confectionary *• products, egg products and pastries and other bakery products. Rhodoionila graci/is CFR 1 is a natural isolate from the Culture Colleelion Centre, Food Microbiology Dept, CFTRI, Mysore. Rliodotornlu grctcilis belonging to the group of imperfect yeast. The cells of Rliotlotorula species are spherical. ovoid or elongate. They reproduce asexually by multilateral budding. It is a oleaginous yeast. C'arotenoid synthesis is a characteristic feature of R.grucilis. It produces a mi\lure of carotenoids p-carotene. lorulene and lorularhodine. The p carotene has attracted commercial interest as a yellow colorant for food in addition to vitamin A precursor. Carotenoids are synthesized in the cytoplasm and always present in the association \\ith lipids. These carotenoids are intracellular and needs breakage of cell walls for the release of carotenoids. The cell wall of R. gracilis is very rigid and consists of 70% of mannose and 20% of glucan and some amount of chitin. For the extraction of carotenoids. normally manual methods are being used to rupture the cell walls. However, these methods are highly laborious and consume a lot of organic soh ent. After harvesting the biomass, the cells are extracted for carotenoids. Normally they are either stored at 4°C or frozen. Under these conditions, if it is stored for a longer time, decolonisation of biomass occurs. When the frozen samples is thawed to room temperature for extraction, loss of pigment oceurs. Hence, in the present invention where in R. graci/is biomass is treated at different temperatures which, prevents the loss of pigment and also the cells can be stored for a longer period of time. A modified of extraction method is used here which is simple and easy. Reference may be made to the Indian pending patent No.382/L)el/2001 where in the extraction of carotenoids is by manual grinding with neutral sand and solvents. Reference may be made to the work carried out by Han R.P , Patel Tand Martin A.M.(1992) A new strain of Rhodotorula rubra isolated from yoghurt.J. Industrial Microbiol.l 1, 43-45, where in the carotenoids was extracted by rupturing the cells in a French press at an internal cell pressure of 32000 psi. Ruptured cells are extracted with acetone and then with petroleum ether 60 -80"C. The drawback of this method is, the extraction is cumbersome using French press and consumption of solvents is more.. Reference may be made to the work carried out by Frengova G,Simova E.Beshkova D,and Grigorova D(1994) Formation of carotenoids by Rhodotonilu glutinis in Whey Ultra 111 irate. Biotechnol Bioeng.44, 888-894. where in, wet cells collected by centrifugalion is subjected to destruction in a Gaulin homogeniser at a temperature of 8 10°C under a pressure of 50 Mpa. Then the carotenoids are extracted with acetone ratio 2:1 for 5 limes. Then again separated with petroleum ether. This method is too lengthv. Reference may be made to the work carried out by Vijayalakshmi G. Shoba B.Vanajakslu V, Divaker S, and Manohar B (2001) Response surface methodology for optimization of growth parameters for the production of carotenoids by a mutant strain of R.gracilis.Eur.Food.Res.Tech .213, 234-239, \\here in the carotenoids are extracted from R.gracilis by grinding the cells \\ith acetone, extracted it with hexane, followed by petroleum ether. In this method, the consumption of solvents is very high and grinding of cells is very tedious job. Exposure to solvents is another problem. Reference may be made to the work carried out by Martin A M, Chun Lu, and Patel RT(1993) Growth parameters for the Yeast Rhodotorula rubra grown in Peat extracts. J. Ferment Bioeng .4, 321-325, where in the yeast cells are ruptured dimethyl sulphoxide, carotenoids were extracted with diethyl ether. The main objective of the present invention is to provide an effective method of retention of carotenoids in Rhodotorula gracilis cells and its extraction which obviates the drawbacks as detailed above . Accordingly, I he present invention provides an effective method of retention of carotenoids in Rhodotorula gracilis cells and its extraction which comprises of - Inoculating of R.gracilis in 1% Enebose medium ,culti\ulmg R.gracilis in 1% Enebose medium for 24 hrs at 28 "('., inoculating 24 hr old culture of R.gracilis into Enebose medium with 4% glucose, cultivating R.gracilis under submerged condition on a rotary shaker (200 rpm) in Enebose medium for 5 days at 28 V, harvesting of R.gracilis by centnfugation at 6000 rpm for 10 minutes., washing of R.gracilis cells with disiilled \\aler twice, spreading of washed cells (with a moisture content of 75%) on to a thin plate to a thickness of less 1mm, exposure of spread cells at 4"C for 24 hrs, collecting of R.gracilis flakes of cells after 24 hrs at 4°C, drying of flakes of R.gracilis at 30°C for 24 hrs, powdering of dry flakes, adding solvents to powered cells, repeated extraction of carotenoids from powdered cells with solvents (3 times), extracting left over carotenoids by incubating powdered, extracted cells with solvents at 28(IC overnight, pooling of extracts, concentrating extracts, measuring of carotenoid extracts spectrophotometrically. In an embodiment of the present invention the medium used for preparation of inoculum is Enebose medium with 1% glucose for 24 hrs. In an another embodiment of the present invention the growth medium for cirlmation R.gracilis is 4% Enebose medium. In yet .mother embodiment of the present invention 10% inoculum is used for the cultixation of R.gracilis by submerged fermentation. In still another embodiment of the present invention R.gracilis is cultivated for 5 days under aerobic condition at 28°C with 200 rpm for carotenoid production. In another embodiment of the present invention, the wet cells are harvested, washed and dried at two different temperatures 4°C followed by 30°C. In another embodiment of the present invention, the dried cells are powdered In another embodiment of the present invention, the powdered cells are extracted with solvents to eel carotenoids. Flow chart for cultivation of' R.gracilis cells Slant % Enebose medium Incubation for 24hr at 28 "C I Inoculum 0% inoculum into growth medium Submerged cultivation of R. gracilis in growth Medium 5 Days Fermented Broth I Harvesting the cells Washing of cells with distilled water Cells ready for extraction of carotenoids Flow chart for extraction of carotenoids from R. gmcilis by modified method Wet cells I Spread on plate as thin layer Left at 4°C for 24 hrs Flakes of R.gracilis cells are collected (4 % moisture) Dried at 30°C for 24 hrs (1% moisture) Powdering of dried cells Dried powdered cells Incubation of cells with acetone at 28°Cfor 1 hr Repetition of the above step three times Decantation of extract Incubation ofresidue with acetone at 28°C for 18h to extract left over carotenoid Pooling of acetone extracts Passing through bed of sodium sulphate Concentration of extracts by flash evaporation Carotenoid extract in chloroform Spectrophotometric measurement Iraditional method ol extraction Wcl cells Washed with water i g wet cells + lOg neutral sand + 20ml acetone Manual grinding Add 15ml of hexane Decant the supernatant Repetition ol'acetone treatment and extraction with hexane once more Residue + 30ml diethyl ether Manual grinding Extraction with diethyl ether Repetition ol'diethyl ether treatment till the cells become colourless Pooling of acetone and diethyl ether extract Passing through bed of sodium sulphate Concentration of ex tract by Hash evaporation Carotenoid extract in chloroform Spectrophotometric measurement Cultivation of R.gracHis: Rhodoioru/d grcicHis CFR 1 was cultivated in 500ml Erlenmeyer llasks containing 100ml of 1 to 2 % Enebose medium, which serves as the initial inoculum. A calculated amount of 20 - 24h old inoculum was inoculated into the llask containing the growth medium. This was incubated on a rotary shaker (150 to250 rpm) for a period of 5 to 7 days at 28 ±2 "C. At the end of fermentation, yeast was harvested by centrifugation (5000-7000 rpm) for 10 lo 15 minutes, washed twice with distilled water (70 to 75% moisture). Extraction of carotenoids: a) Treatment of cells at low temperature: The washed cells were spread as a thin layer on a petriplate and is kept at 4(IC overnight (20 to 24 hrs) where in the colour of the cells are intensified. The moisture content of the cells is around 3 to 5 % b) Treatment of cells at higher temperature: The Hakes of R. gracilis cells are further dried at28 to 32 "C for 24 hrs to a moisture content of 1 to 2 %. c) Powdering of R. gracilis: llie dried cells are collected and ground into a fine powder using a dry m'inder. d) Extraction of total carotenoids: Known quantity of fine powder is taken in a test tube /IIask to which 10ml of solvent is added, left at room temperature for 1 hr. This solvent treatment was repeated 3 times at an interval of 1 hr, and then left at 28 t 2 "C over night for 18 to 24 hr at 30 tO 32°C. Maximum amount of carotenoid is extracted by then. All the concerned solvent extracts are pooled together and passed through a bed of anhydrous sodium sulphate. This clear carotenoid extract was flash evaporated at 30 to 35(IC with 120 rpm under 500 IBC vacuum to dryness and slowly to 350 IBC vaccum.The extract is taken in 10 to 2o ml of chloroform for estimation. e) Estimation of carotenoids: The carotenoid content were estimated by absorption spectrum of the carotenoid solution at 460 nm. A molar extinction coefficient value of ()9330cm 1 mol obtained from an average value of E from ten carotenoids (lycopene, a - carotene, p1- carotene, 8- carotene, Zeaxanthin, rhodoxanthm, astaxanthin. Lutein. p- apo-2 carotenal dihydro P-carotene ) was used in the following equation for determining the percentage of carotenoid present in the sample. Percentage of carotenoids = 5.405 x 10"4 x (A460 X/Y), where A460 is the absorbance of X ml of solution obtained from Y g of dry cells. The dry weight of the cells employed was determined from the estimated moisture content of the wet cells. Ultraviolet visible spectroscopic measurement were earned out using a IIV-1 60 recording Spectrophotometer (Shimad/u. Japan). Example 1 1.0 g of wet cells are taken in a pestle and mortar. Two spatula (10 g) lull of acid and alkali washed sand (neutral sand) was added to the cells. 20 ml of acetone was added to the mixture and ground with the pestle. Then 15 ml of hexane is added and the extract is collected in the cylinder. Another 20 ml of acetone is added to the cells, ground.to a fine mass. 15 ml of hexane is added and the extract is decanted into the cylinder. The cells are further extracted with 30 ml of diethyl ether. The cells are repeatedly extracted with diethyl ether till the cells become colourless. All the solvents extracts-are pooled together passed through a bed of anhydrous sodium sulphate, concentrated by Hash evaporation, made up to known volume with chloroform and measured specirophotometncally. The total carotenoid yield is 400 (.ig/gdcm. Example 2 O.lg of powdered R.gracilis cells was taken into a test lube. 10ml of acetone was added, mixed thoroughly and allowed to stand at room temperature for 1 hour. After Ih, the supernatant is decanted/ centrifuged. To the residue, 10ml of acetone was added and kept at room temperature for another hour. This treatment was repeated three times. After treating the residue with acetone three times, the residue was left overnight at room temperature (28"C) with 10ml of acetone. Next day the supernatant is decanted. All the previous acetone extracts are pooled together, concentrated by flash evaporation, made up to known volume with chloroform and measured spectrophotometncally. The total carotenoid yield is 344 jag/gdcm. Example 3 O.lg of powdered R.gracilis cells was taken into a test tube. 10ml of methanol \\as added mixed thoroughly and allowed to stand at room temperature for 1 hour. After Ih, the supernatant is decanted/ centrifuged. To the residue, 10ml of melhanol was added and kept at room temperature for another hour. This treatment was repeated three times. After treating the * residue with methanol three limes, the residue was left overnight at room temperature (28UC) with 10ml of methanol. Next day the supernatant isdecanted. All the previous methanol extracts are pooled together, concentrated, b\ Hash evaporation, made up to known volume with chloroform and measured spectrophotometrically. The total carotenoid yield is 260 )Lig/gdcm. Example 4 O.lg of powdered R.gracilis cells was taken into a test tube. 10ml of ethanol was added mixed thoroughly and allowed to stand at room temperature for I hour. After Ih, the supernatant is decanted/ centrifuged. To the residue, 10ml of ethanol was added and kept at room temperature for another hour. This treatment was repeated three times. After treating the residue with ethanol three times, the residue was left overnight at room temperature (28°C) with 10ml of ethanol. Next day the supernatant is decanted. All the previous ethanol extracts are pooled together, concentrated, by flash evaporation, made up to known volume with chloroform and measured spectrophotometrically. The total carotenoid yield is 93 j.ig/gdcm. Example 5 O.lg of powdered R.gracilis cells was taken into a test tube. 10ml of propanol was added mixed thoroughly and allowed to stand at room temperature tor 1 hour. After Ih, the supernatant is decanted/ centrifuged. To the residue, 10ml of propanol was added and kept at room temperature for another hour. This treatment was repeated three times. After treating the residue \\ith propanol three times, the residue was left o\ernight at room temperature (28l)C) with 10ml of propanol. Next day the supernatant is decanted. All the previous propanol extracts are pooled together, concentrated, by flash evaporation, made up to known volume with chloroform and measured spectrophotometrically. The total carotenoid yield is 234 ug/gdcm. Example 6 O.lg of powdered R.gmcilis cells was taken into a lest tube. 10ml of chloroform was added mixed thoroughly and allowed to stand at room temperature for 1 hour. After Ih, the supernatant is decanted/ centrifuged. To the residue, 10ml of chloroform was added and kept at room temperature «•• for another hour This treatment was repeated three times. After treating the residue with chloroform three times, the residue was left overnight at room mperature (28'C) with 10ml of chloroform. Next day the supernatant is decanted. All the previous chloroform extracts are pooled together, concentrated, by flash evaporation, made up to known volume with chloroform and measured spectrophotometncally. The total carotenoid yield is 144 u,g/gdcm. Ex ample 7 O.lg of powdered R.gracilis cells was taken into a test tube. 10ml of hexane was added mixed thoroughly and allowed to stand at room temperature for 1 hour. After Ih, the supernatant is decanted/ centrifuged. To the residue, 10ml of hexane was added and kept at room temperature for another hour. This treatment was repeated three times. After treating the residue with hexane three times, the residue was left overnight at room temperature (28II( ) with 10ml of hexane. Next day the supernatant is decanted. All the previous hexane extracts are pooled together, concentrated, b\ flash evaporation, made up to known volume with chloroform and measured speclrophotomeincally. The total carotenoid yield is 23 lu.g/gdcm. Bxample 8 0. Ig of powdered R.grucilis cells was taken into a test tube. 10ml ofdiethyl ether was added mixed thoroughly and allowed to stand at room temperature for I hour. After 1 h, the supernatant is decanted/ centrifuged. To the residue. 10ml ofdiethyl ether was added and kept at room temperature for another hour. This treatment was repeated three times. After treating the residue with diethyl ether three times, the residue was left overnight at room temperature (28°C) with 10ml ofdiethyl ether. Next day the supernatant is decanted. All the previous diethyl ether extracts are pooled together, concentrated, by Hash evaporation, made up to known volume with chloroform and measured speetrophotomeincally. The total carotenoid yield is 223 u.g/gdcm. Example 9 O.lg of powdered R.gracilis cells was taken into a test tube. 10ml of petroleum ether was added mixed thoroughly and allowed to stand at room temperature for 1 hour. After In, the supernatant is decanted/ centrifuged. To the residue, 10ml of petroleum ether was added and kept at room temperature for another hour. This treatment was repeated three times. After treating the residue with petroleum ether three times, the residue was left overnight at room temperature (28()C) with 10ml of petroleum ether. Next day the supernatant is decanted. All the previous petroleum ether extracts are pooled together, concentrated, by Hash evaporation, made up to known volume with chloroform and measured spectrophotometrically. The total carotenoid yield is 242 f.ig/gdcm. The mam advantages ofthe present invention are 1. No manual grinding of cells with solvents is involved 2. Amount of solvent used is less (50ml) for O.lg of dry cells as compared to 200ml for Ig wet cells (0.25g dry cells). 3. Ir.xposure of cells to 4° C reduces the loss of colour. 4. This helps storing of R..gracilis cells for longer period of time without loss of pigment. 5. Physical exposure to solvent is minimized. 6. Simple and easy method. We claim: 1. An improved process for an extraction of carotenoids from Rhodotorula gracilis cells and its extraction which comprises: a) cultivating R.gracilis in 1% Enebose medium for 24 hrs at 26-28°C with a pH range of 5-5.5 b) inoculating the above culture of R. Gracilis into Enebose medium with 4-10% glucose with 106 - 108 cells/ml of R. Gracilis c) cultivating R. Gracilis at 200 rpm in Enebose medium for 5 days at 26-28°C d) harvesting of R. Gracilis by Centrifugation at 6000 rpm for 10 minutes e) washing of the above culture two times with distilled water f) spreading of washed cells with a moisture content of 75% on to a thin plate to a thickness of less than 1 mm g) exposing the spread cells in light at 4°C for 24 hrs to obtain flakes of R Gracilis h) drying the above said flakes at 30°C for 24 hrs i) powdering of dry flakes using a dry grinder. j) adding lOmL of acetone to 0.1 g of R.gracilis cells . k)mixing thoroughly and allowing it to stand at room temperature for lhr. 1) decanting the supernatant, adding solvents such as herein described and keeping it at room temperature for 1 hr. m) treating the residue with solvents such as herein described three times. n) keeping the residue with 10 mL solvent overnight at 28°C. o) decanting the supernatant. p) concentrating the residue by flash evaporation, made upto known volume with chloroform and measured spectrophotometrically. 2. A process claimed in claim 1, wherein the organic solvent used in step (1) and (m) is selected from the group consisting of methanol, ethanol, acetone, propanol, and chloroform, hexane, diethyl ether, petroleum ether. 3. An improved process for extraction of carotenoids substantially as herein described with reference to the examples

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420-del-2003-abstract.pdf

420-DEL-2003-Claims-(03-12-2008).pdf

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420-del-2003-description (complete).pdf

420-del-2003-form-1.pdf

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