Title of Invention	A PROCESS FOR THE PRODUCTION OF CAROTENOID FROM MICROBIAL SOURCE USING WHEAT BRAN EXTRACT
Abstract	Present invention provides a process for the production of carotenoids from microbial source using wheat bran extract by preparing wheat bran extract by soaking, extracting followed by filtering of the wheat bran with water, at 20 - 97°C temperature and enriching of the above filtrate with carbon source at 1.5 to 4.0% level and pH of 4.5 to 6.0m and culturing yeast cell suspension in this wheat bran extract aerobically for 36 to 168 hr in a state of rotary motion at 25 - 32°C, followed by harvesting the above cell biomass from fermented broth for extraction of carotenoid by using an organic solvents.

Title of Invention

A PROCESS FOR THE PRODUCTION OF CAROTENOID FROM MICROBIAL SOURCE USING WHEAT BRAN EXTRACT

Abstract

Present invention provides a process for the production of carotenoids from microbial source using wheat bran extract by preparing wheat bran extract by soaking, extracting followed by filtering of the wheat bran with water, at 20 - 97°C temperature and enriching of the above filtrate with carbon source at 1.5 to 4.0% level and pH of 4.5 to 6.0m and culturing yeast cell suspension in this wheat bran extract aerobically for 36 to 168 hr in a state of rotary motion at 25 - 32°C, followed by harvesting the above cell biomass from fermented broth for extraction of carotenoid by using an organic solvents.

Full Text	The present invention relates to a process for the production of carotenoid from microbial source. Carotenoids are required as feed supplements in the poultry industry and in aquaculture of fishes and crustaceans. Besides, providing nutrition and possibly disease resistance, carotenoids give brilliant pigmentation and aesthetic value to Crustacea, animals, etc. Recent studies have suggested that carotenoids have health benefits in humans and animals by preventing or delaying some chronic diseases including cancer, arteriosclerosis and cataract. These findings have contributed greatly to the industrial and medical interests in carotenoids. Most of the carotenoids used in the food, feed, nutritional supplement, cosmetic, and pharmaceutical industries or for other industrial purposes are produced by total chemical synthesis. Production of natural food colours is gaining importance throughout the world, due to increase in public awareness and also due to the growing consumer demand for safe, natural food colourants. Because of technological limitations of synthetic colourants, there is a growing interest in the development and application of colourants from microbial sources. Certainly, a primary contributing factor to the increased interest in natural carotenoids is the current trend of avoiding food additives and synthetic chemicals in foods. Microbial carotenoids have attracted much interest in recent years for several reasons, the primary reason is the growth of aquaculture - currently the fastest growing sector of agriculture. Astaxanthin is an important source of pigmentation, p-carotene, on the other hand, is an important food colourant as well as the source of vitamin-A in animal diets. The market for these two carotenoids is rapidly growing and only microbial synthesis can meet the demand for biological carotenoids. The rapid growth of microbes reduces the production time to a matter of days and the process lends itself to a continuous operation. Carotenoid pigment biosynthesis is a characteristic ability of genus Rhodotorula. It produces a mixture of carotenoids such as P-carotene, torulene and torularhodine. The P-carotene found in this yeast has attracted commercial interest as a precursor of vitamin-A and as a yellow pigment for food. Reference may be made to the work carried out by Frengova et al. (1994) wherein whey filtrate was used as a substrate for formation of carotenoids by R.gracilis with co-cultivation of homo-fermentative lactic acid bacteria. The draw back in this method is a synthetic medium consisting of high concentration of glucose, yeast extract, phosphate and magnesium sulphate as growth medium which is expensive and also separation of carotenoids from a mixed culture is not suitable. Reference may be made to Martin et al. (1993) wherein acid extracts of peat was used as the main substrate source for carotenoids production by Rhodotorula rubra. The draw back of this method is the supplementation of nutrients such as (NH4)2S04, yeast extract, K2SO4 and MgSO4.7H2O. Carotenoid concentration reported is 1,256 ug/g dry mass. Peat is not a commonly available substrate for use. Reference may be made to Buzzini and Martini (1999) wherein grape must, glucose syrup, beet molasses, soyabean flour extract maize flour extract, etc were used individually as carbon substrate for carotenoid production. In these media, other nutrients such as glucose, KH2P04, MgS04.7H2O, yeast extract were also used. The total carotenoid reported with (a) concentrated grape must is 915.4 µg/g dry cells, (b) grape must 455.4 µg/g, (c) glucose syrup 240 µg/g, (d) beet (e) molasses 332.8 µg/g, (e) soyabean flour extract 883.3µg/g and with maize flour extract 383.9 µg/g dry cells only. Reference may be made to the work carried out by Costa et al. (1987), wherein a chemical defined medium consisting of sucrose, (NH4)2SO4, KH2PO4, Na2HPO4, MgSO4.7H2O yeast extract, with pH 6.5 was used and 605 ug/g of dry cells of P-carotene is obtained when cells are maintained under non-proliferating conditions. This process involves an additional step of harvesting, washing and second stage of fermentation. Reference may be made to the work carried out by Shih and Hang (1996), wherein sauerkraut brine is used as a substrate for production of carotenoids and the maximum yield of total carotenoids expressed as P-carotene was only 16.35 ug/g yeast on dry weight basis. The technical know how of using wheat bran extract for carotenoid production under submerged fermentation is not established so far. The main object of the present invention is a process for the production of carotenoid from microbial source. Accordingly, present invention provides a process for the production of carotenoids from microbial source using wheat bran extract, which comprises: a) soaking, extracting followed by filtering of the wheat bran with water, at 20 - 97°C temperature, b) enriching of the above filtrate with carbon source such as herein described at 1.5 to 4.0% level and pH of 4.5 to 6.Om c) sterilizing the enriched extract at 115 - 125°C for 15 to 25 minutes followed by cooling to 27 to 32°C, d) inoculating the above sterilized extract with 8 to 12% yeast cell suspension such as defined herein , e) fermenting aerobically for 36 to 168 hr in a state of rotary motion at 25 - 32°C, f) harvesting the above cell biomass from fermented broth for extraction of carotenoid by using an organic solvents. In embodiment of the present invention extraction may be carried out with water at 20 to 97°C temperature. In another embodiment of the present invention, the carbon source used for enrichment may be glucose at 1.5 to 4% maximum at pH 4.5 to 6.0. In an embodiment of the present invention, the sterilization may be carried out at 120 - 122°C for 20 to 22 minutes. In an another embodiment, the yeast cell suspension may contain Rhodotorula gracilis at 8 - 12 % concentration. Rhodotorula gracilus (ATCC 90950) produces a mixture of carotenoids such as torulene, torulahodine and beta-carotene which find its application as food colourant in cosmetics, Pharmaceuticals and also in poultry feed. The yeast in use produces more of beta-carotene. In addition, the yeast also produces lipids which may add value to the product. In another embodiment of the present invention, the cell suspension of Rhodotorula gracilis may be 20 to 24 hr old culture. In an another embodiment, the broth may be fermented aerobically for a period of 36 - 168 hr. In an another embodiment of the invention, the solvents used for extraction may be from acetone, hexane and diethyl ether. In an embodiment of the invention the yield of total carotenoid may be 1100 to 1300 µg/g dry cell mass. The detailed process for the production of carotenoid is presented below along with the flow diagram. The detail process for production of carotenoid is as follows: R.gracilis CFR1 was maintained on PDA agar (slants) consisting per litre glucose 20.Og, potato infusion from 200g of potato, agar 20.0g at 4°C. Preparation of wheat bran extract medium l.Og of wheat bran was taken in a beaker, suspended in 50 ml of distilled water, left at ambient temperature for a period of one hour. After one hour of soaking, the wheat bran extract was filtered through a muslin cloth and centrifuged. The supernatant was collected in a conical flask. The residue was washed 3 times with water. The supernatant are pooled together and the residue discarded. To the filtrate 2 g of glucose was added. The volume was made up to 85 ml. pH was adjusted to 5.5 with 0.1N NaOH/O.lN HC1. The final volume was made upto 92 ml. The medium was sterilised at 121°C for 20 min, cooled and used as medium for production of carotenoids. Preparation of inoculum 1% Enebose medium was used as growth medium for inoculum preparation. The following ingredients glucose 1 g, CaCl2.2H2O (0.04g), MgSO4.7H2O (0.04g), yeast extract (0.15 g), NH4N03 (0.0286 g), KH2PO4 (0.075 g) were weighed in a beaker, dissolved completely in 95 ml of water. pH was adjusted to 5.5 with 0.1N NaOH/O.lN HC1. The final volume was made up to 100 ml. This medium was taken in a 500 ml Erlenmeyer flasks, sterilised at 121°C for 20 min, cooled to room temperature. A loop of 3 days old culture of R.gracilis from PDA slants was transferred into Enebose medium with 1% glucose, incubated on a rotary shaker (200 rpm) at ambient temperature (28+2°C) for 20 hr. At the end of 20 hr, 10 ml of this cell suspension (fermented broth) was used for inoculation of wheat bran medium for carotenoid production. Cultivation of yeast cells for carotenoid production 10 ml of 20 hr old cell culture of R.gracilis (107/ml) was inoculated into wheat bran medium (90 ml). This was incubated on a rotary shaker (200 rpm) at ambient temperature 28+2°C for 120 hours. Harvesting At the end of fermentation, yeast cells were harvested by centrifugation (6000 rpm) for 15 min. The supernatant was discarded. The cells were washed twice with distilled water to remove excessive nutrients. This biomass was used for extraction of carotenoids. Extraction of carotenoids was carried out as follows: 1.0 g of wet cells are taken in a pestle and mortar. Two spatula (10 g) full 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 a cylinder. Another 20 ml of acetone was added to the cells, ground to 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 solvent extracts are pooled together passed through a bed of anhydrous sodium sulphate. This clear carotenoid extract was flash evaporated at 35°C with 120 rpm under 200 Ib vacuum to dryness. The extract is taken in 10 ml of chloroform for estimation. Estimation of carotenoids The carotenoid content were estimated by absorption spectrum of the carotenoid solution at 460 nm. A molar extinction coefficient value of 99330 cm/l/mol obtained from an average value of E from ten carotenoids (lycopene, alpha-carotene, ß-carotene, δ-carotene, zeaxanthin, rhodoxanthin, astaxanthin, lutein, ß-apo-2 carotenal and dihydro ß-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 measurements were carried out using a UV-160 recording Spectrophotometer (Shimadzu, Japan). In the above process, other nutrients such as yeast extract, MgSO4. 7H2O, KH2PO4, NH4NO3 and CaCl2.H2O are eliminated. All these are replaced by just the water extract of wheat bran which was later enriched with glucose for the growth of the yeast for carotenoid production. Flow diagram for the process Wheat bran Water retraction Extract + Wg of glucose pH adjusted to 5.5 Sterilised using autoclave Inoculated with N.gracilis cells 10% Fermentation for 120hr at room temperature Harvest cellusing centrifuge Cells washedwith plain water Extracted with solvents (hexane, acetone, diethyl ether) imnatic Concentrate and estimation of carotenoids Novelty of the process 1. Use of wheat bran as substrate 2. Higher yield of carotenoid compared to any reported literature 3. Use of microbial source EXAMPLE 1 0.5 g of wheat bran was taken in a beaker and was suspended in 50 ml of distilled water, left at 20°C for a period of one hour. After one hour of soaking, the mixture (wheat bran + water) was filtered through a muslin cloth and then centrifuged. The centrifuged filtrate was collected in a conical flask. The residue was washed 3 times with 10 ml of distilled water each. The filtrates are pooled together and the residue was discarded. To the filtrate, the following ingredients: glucose (4.0g), CaCl2.H2O (0.04 g), MgSO4.7H2O (0.04 g), KH2PO4 (0.075 g) and NH4NO3 (0.0286 g) were added, dissolved completely by mixing. The volume was made up to 85 ml, pH was adjusted to 5.5 with 0.1N NaOH/O.lN HC1. The final volume was made upto 92 ml. The medium was sterilised at 121°C for 20 minutes, cooled to 28+2°C. The sterilised extract with ingredients was inoculated with 10% of 16 hr old cell suspension ofR.gracilis under sterile condition. This was incubated on a rotary shaker (200 rpm) at 25°C for a period of 144 hrs. Later the biomass is harvested by centrifugation (6000 rpm) washed with water twice and taken for extraction of carotenoids. The total carotenoid yield is 510 µg/g dcm. EXAMPLE 2 1.0 g of wheat bran was taken in a beaker and was suspended in 50 ml of distilled water, left at 28°C for a period of one hour. After one hour of soaking, the mixture (whet bran + water) was filtered through a muslin cloth and centrifuged. The centrifuged filtrate was collected in a conical flask. The residue was washed 3 times with 10 ml of distilled water. The filtrates are pooled together and the residue was discarded. To the filtrate, the following ingredients: glucose (4.0g), CaCl2.H2O (0.04 g), MgSO4.7H2O (0.04 g), KH2PO4 (0.075 g) and NH4NO3 (0.0286 g) were added, dissolved completely by mixing. The volume was made up to 85 ml, pH was adjusted to 5.5 with 0.1N NaOH/O.lN HC1. The final volume made upto 92 ml. The medium was sterilised at 121°C for 20 minutes, cooled to 28±2°C. The sterilised extract with ingredients was inoculated with 10% of 20 hr old cell suspension ofR.gracilis under sterile condition. This was incubated on a rotary shaker (200 rpm) at 28°C for a period of 144 hrs. Later the biomass is harvested by centrifugation (6000 rpm) washed with water twice and taken for extraction of carotenoids. The total carotenoid yield is 630µg/g dcm. EXAMPLES 2.0 g of wheat bran was taken in a beaker and was suspended in 50 ml of distilled water, left at 32°C for one hour. After one hour of soaking, the mixture (whet bran + water) was filtered through a muslin cloth and centrifuged. This centrifuged filtrate was collected in a conical flask. The residue was washed 3 times with 10 ml of distilled water. The filtrates are pooled together and the residue was discarded. To the filtrate, the following ingredients: glucose (4.0g), CaCl2.H2O (0.04 g), MgSO4.7H2O (0.04 g), KH2P04 (0.075 g) and NH4NO3 (0.0286 g) were added, dissolved completely by mixing. The volume was made up to 85 ml, pH was adjusted to 5.5 with O.IN NaOH/O.lN HCI. The final volume made upto 92 ml. The medium was sterilised at 121°C for 20 minutes, cooled to 28+2°C. The sterilised extract with ingredients was inoculated with 10% of 16 hr old cell suspension ofR.gracilis under sterile condition. This was incubated on a rotary shaker (200 rpm) at 32°C for a period of 144 hrs. Later the biomass is harvested by centrifugation (6000 rpm) washed with water twice and taken for extraction of carotenoids. The total carotenoid yield is 410 jig/g dcm. EXAMPLE 4 0.5 g of wheat bran was taken in a beaker. 60 ml of distilled water was added and boiled (97°C) on a slow flame for 15 minutes, cooled and filtered through a muslin cloth and centrifuged. The centrifuged filtrate was collected in a conical flask. The cooled residue was further washed 2 times with 10 ml of water. All the filtrates were pooled together and the residue was discarded. To the filtrate, the following ingredients: glucose (4.0g), CaCl2.H2O (0.04 g), MgSO4.7H20 (0.04 g), KH2P04 (0.075 g) and NH4NO3 (0.0286 g) were added, dissolved completely by mixing. The volume was made up to 85 ml, PH was adjusted to 5.5 with 0.1N NaOH/O.lN HC1. The final volume made upto 92 ml. The medium was sterilised at 121°C for 20 minutes, cooled to 28±2°C. The sterilised extract with ingredients was inoculated with 10% of 24 hr old cell suspension ofR.gracilis under sterile condition. This was incubated on a rotary shaker (200 rpm) at 28°C for a period of 120 hrs. Later the biomass is harvested by centrifugation (6000 rpm) washed with water twice and taken for extraction of carotenoids. The total carotenoid yield is 540 µg/g dcm. EXAMPLES 1.0 g of wheat bran was taken in a beaker. 60 ml of distilled water was added and boiled (97°C) on a slow flame for 15 minutes, cooled and filtered through a muslin cloth and centrifuged. The centrifuged filtrate was collected in a conical flask. The cooled residue was further washed 2 times with 10 ml of water. All the filtrates were pooled together and the residue was discarded. To the filtrate, the following ingredients: glucose (4.0g), CaCl2.H20 (0.04 g), MgSO4.7H2O (0.04 g), KH2PO4 (0.075 g) and NH4NO3 (0.0286 g) were added, dissolved completely by mixing. The volume was made up to 85 ml, pH was adjusted to 5.5 with 0.1N NaOH/O.lN HC1. The final volume made upto 92 ml. The medium was sterilised at 121°C for 20 minutes, cooled to 28±2°C. The sterilised extract with ingredients was inoculated with 10% of 20 hr old cell suspension ofR.gracilis under sterile condition. This was incubated on a rotary shaker (200 rpm) at 28°C for a period of 144 hrs. Later the biomass is harvested by centrifugation (6000 rpm) washed with water twice and taken for extraction of carotenoids. The total carotenoid yield is 522 ug/g dcm. EXAMPLE 6 2.0 g of wheat bran was taken in a beaker. 60 ml of distilled water was added and boiled on a slow flame for 15 minutes (97°C), cooled and filtered through a muslin cloth and centrifuged. The centrifuged filtrate was collected in a conical flask. The cooled residue was further washed 2 times with 10 ml of water. All the filtrates were pooled together and the residue was discarded. To the filtrate, the following ingredients: glucose (4.0g), CaCl2.H2O (0.04 g), MgSO4.7H2O (0.04 g), KH2PO4 (0.075 g) and NH4NO3 (0.0286 g) were added, dissolved completely by mixing. The volume was made up to 85 ml, pH was adjusted to 5.5 with 0.1N NaOH/O.lN HC1. The final volume made upto 92 ml. The medium was sterilised at 121°C for 20 minutes, cooled to 28±2°C. The sterilised extract with ingredients was inoculated with 8% of 20 hr old cell suspension of R.gracilis under sterile condition. This was incubated on a rotary shaker (200 rpm) at 28°C for a period of 120 hrs. Later the biomass is harvested by centrifugation (6000 rpm) washed with water twice and taken for extraction of carotenoids. The total carotenoid yield is 470 µg/g dcm. EXAMPLE 7 1.0 g of wheat bran was taken in a beaker, and suspended in 50 ml of distilled water. Left at 28°C for a period of one hour. After one hour of soaking, the mixture (wheat bran + water) was filtered through a muslin cloth and then centrifuged. The centrifuged filtrate was collected in a conical flask. The residue was washed 3 times with 10 ml of distilled water. The filtrates were pooled together and the residue was discarded. To the filtrate, 1.5 g of glucose was added, dissolved completely by mixing. The volume is made upto 85 ml, pH was adjusted to 5.5 with 0.1N NaOH/O.lN HC1. The final volume was made upto 92 ml. The medium was sterilised at 121°C for 20 minutes and cooled. The sterilised extract with glucose was inoculated with 10% of 20 hr old culture suspension of R.gracilis under sterile condition. This was incubated on a rotary shaker (200 rpm) at 28°C for a period of 120 hrs. Later the biomass was harvested by centrifugation (6000 rpm) washed with distilled water twice and taken for extraction of carotenoids. The total carotenoid yield is 1100 µg/g dcm. EXAMPLE 8 1.0 g of wheat bran was taken in a beaker, and suspended in 50 ml of distilled water. Left at 28°C for a period of one hour. After one hour of soaking, the mixture (wheat bran + water) was filtered through a muslin cloth and then centrifuged. The centrifuged filtrate was collected in a conical flask. The residue was washed 3 times with 10 ml of distilled water. The filtrates were pooled together and the residue was discarded. To the filtrate, 3 g of glucose was added, dissolved completely by mixing. The volume is made upto 85 ml, pH was adjusted to 5.5 with 0.1N NaOH/O.lN HC1. The final volume was made upto 92 ml. The medium was sterilised at 121°C for 20 minutes and cooled. The sterilised extract with glucose was inoculated with 10% of 20 hr old culture suspension of R.gracilis under sterile condition. This was incubated on a rotary shaker (200 rpm) at 28°C for a period of 120 hrs. Later the biomass was harvested by centrifugation (6000 rpm) washed with distilled water twice and taken for extraction of carotenoids. The total carotenoid yield is 800 µg/g dcm. EXAMPLE 9 1.0 g of wheat bran was taken in a beaker, and suspended in 50 ml of distilled water. Left at 28°C for a period of one hour. After one hour of soaking, the mixture (wheat bran + water) was filtered through a muslin cloth and then centrifuged. The centrifuged filtrate was collected in a conical flask. The residue was washed 3 times with 10 ml of distilled water. The filtrates were pooled together and the residue was discarded. To the filtrate, 4 g of glucose was added, dissolved completely by mixing. The volume is made upto 85 ml, pH was adjusted to 5.5 with 0.1N NaOH/O.lN HC1. The final volume was made upto 92 ml. The medium was sterilised at 121°C for 20 minutes and cooled. The sterilised extract with glucose was inoculated with 10% of 20 hr old culture suspension of R.gracilis under sterile condition. This was incubated on a rotary shaker (200 rpm) at 28°C for a period of 120 hrs. Later the biomass was harvested by centrifugation (6000 rpm) washed with distilled water twice and taken for extraction of carotenoids. The total carotenoid yield is 900µg/g dcm. EXAMPLE 10 1.0 g of wheat bran was taken in a beaker, and suspended in 50 ml of distilled water. Left at 28°C for a period of one hour. After one hour of soaking, the mixture (wheat bran + water) was filtered through a muslin cloth and then centrifuged. The centrifuged filtrate was collected in a conical flask. The residue was washed 3 times with 10 ml of distilled water. The filtrates were pooled together and the residue was discarded. To the filtrate, 2 g of glucose was added, dissolved completely by mixing. The volume is made upto 85 ml, pH was adjusted to 4.5 with O.IN NaOH/O.lN HCI. The final volume was made upto 92 ml. The medium was sterilised at 121°C for 20 minutes and cooled. The sterilised extract with glucose was inoculated with 10% of 20 hr old culture suspension of R.gracilis under sterile condition. This was incubated on a rotary shaker (200 rpm) at 28°C for a period of 120 hrs. Later the biomass was harvested by centrifugation (6000 rpm) washed with distilled water twice and taken for extraction of carotenoids. The total carotenoid yield is 1000 µg/g dcm. EXAMPLE 11 1.0 g of wheat bran was taken in a beaker, and suspended in 50 ml of distilled water. Left at 28°C for a period of one hour. After one hour of soaking, the mixture (wheat bran + water) was filtered through a muslin cloth and then centrifuged. The centrifuged filtrate was collected in a conical flask. The residue was washed 3 times with 10 ml of distilled water. The filtrates were pooled together and the residue was discarded. To the filtrate, 2.0 g of glucose was added, dissolved completely by mixing. The volume is made upto 85 ml, pH was adjusted to 5.5 with O.IN NaOH/O.lN HCI. The final volume was made upto 92 ml. The medium was sterilised at 121°C for 20 minutes and cooled. The sterilised extract with glucose was inoculated with 10% of 20 hr old culture suspension of R.gracilis under sterile condition. This was incubated on a rotary shaker (200 rpm) at 28°C for a period of 120 hrs. Later the biomass was harvested by centrifugation (6000 rpm) washed with distilled water twice and taken for extraction of carotenoids. The total carotenoid yield is 1300 µg/g dcm. EXAMPLE 12 1.0 g of wheat bran was taken in a beaker, and suspended in 50 ml of distilled water. Left at 28°C for a period of one hour. After one hour of soaking, the mixture (wheat bran + water) was filtered through a muslin cloth and then centrifuged. The centrifuged filtrate was collected in a conical flask. The residue was washed 3 times with 10 ml of distilled water. The filtrates were pooled together and the residue was discarded. To the filtrate, 2.0 g of glucose was added, dissolved completely by mixing. The volume is made upto 85 ml, pH was adjusted to 6.0 with 0.1N NaOH/O.lN HCI. The final volume was made upto 92 ml. The medium was sterilised at 121°C for 20 minutes and cooled. The sterilised extract with glucose was inoculated with 10% of 20 hr old culture suspension of R.gracilis under sterile condition. This was incubated on a rotary shaker (200 rpm) at 28°C for a period of 168 hrs. Later the biomass was harvested by centrifugation (6000 rpm) washed with distilled water twice and taken for extraction of carotenoids. The total carotenoid yield is 720 µg/g dcm. EXAMPLE 13 1.0 g of wheat bran was taken in a beaker, and suspended in 50 ml of distilled water. Left at 28°C for a period of one hour. After one hour of soaking, the mixture (wheat bran + water) was filtered through a muslin cloth and then centrifuged. The centrifuged filtrate was collected in a conical flask. The residue was washed 3 times with 10 ml of distilled water. The filtrates were pooled together and the residue was discarded. To the filtrate, 1.5 g of glucose was added, dissolved completely by mixing. The volume is made upto 85 ml, pH was adjusted to 5.5 with 0. IN NaOH/0. IN HCI. The final volume was made upto 92 ml. The medium was sterilised at 121°C for 20 minutes and cooled. The sterilised extract with glucose was inoculated with 6% of 20 hr old culture suspension of R.gracilis under sterile condition. This was incubated on a rotary shaker (200 rpm) at 28°C for a period of 120 hrs. Later the biomass was harvested by centrifugation (6000 rpm) washed with distilled water twice and taken for extraction of carotenoids. The total carotenoid yield is 937 µg/g dcm. EXAMPLE 14 1.0 g of wheat bran was taken in a beaker, and suspended in 50 ml of distilled water. Left at 28°C for a period of one hour. After one hour of soaking, the mixture (wheat bran + water) was filtered through a muslin cloth and then centrifuged. The centrifuged filtrate was collected in a conical flask. The residue was washed 3 times with 10 ml of distilled water. The filtrates were pooled together and the residue was discarded. To the filtrate, 2.0 g of glucose was added, dissolved completely by mixing. The volume is made upto 85 ml, PH was adjusted to 5.5 with O.IN NaOH/O.lN HCI. The final volume was made upto 92 ml. The medium was sterilised at 121°C for 20 minutes and cooled. The sterilised extract with glucose was inoculated with 12% of 20 hr old culture suspension of R.gracilis under sterile condition. This was incubated on a rotary shaker (200 rpm) at 28°C for a period of 120 hrs. Later the biomass was harvested by centrifugation (6000 rpm) washed with distilled water twice and taken for extraction of carotenoids. The total carotenoid yield is 1170 µg/g dcm. EXAMPLE 15 1.0 g of wheat bran was taken in a beaker, and suspended in 50 ml of distilled water. Left at 28°C for a period of one hour. After one hour of soaking, the mixture (wheat bran + water) was filtered through a muslin cloth and then centrifuged. The centrifuged filtrate was collected in a conical flask. The residue was washed 3 times with 10 ml of distilled water. The filtrates were pooled together and the residue was discarded. To the filtrate, 2.0 g of glucose was added, dissolved completely by mixing. The volume is made upto 85 ml, pH was adjusted to 5.5 with O.IN NaOH/O.lN HCI. The final volume was made upto 92 ml. The medium was sterilised at 121°C for 20 minutes and cooled. The sterilised extract with glucose was inoculated with 10% of 20 hr old culture suspension of R.gracilis under sterile condition. This was incubated on a rotary shaker (200 rpm) at 28°C for a period of 36 hrs. Later the biomass was harvested by centrifugation (6000 rpm) washed with distilled water twice and taken for extraction of carotenoids. The total carotenoid yield is 43 µg/g dcm. EXAMPLE 16 1.0 g of wheat bran was taken in a beaker, and suspended in 50 ml of distilled water. Left at 28°C for a period of one hour. After one hour of soaking, the mixture (wheat bran + water) was filtered through a muslin cloth and then centrifuged. The centrifuged filtrate was collected in a conical flask. The residue was washed 3 times with 10 ml of distilled water. The filtrates were pooled together and the residue was discarded. To the filtrate, 2.0 g of glucose was added, dissolved completely by mixing. The volume is made upto 85 ml, pH was adjusted to 5.5 with 0.1N NaOH/O.lN HC1. The final volume was made upto 92 ml. The medium was sterilised at 121°C for 20 minutes and cooled. The sterilised extract with glucose was inoculated with 10% of 20 hr old culture suspension of R.gracilis under sterile condition. This was incubated on a rotary shaker (200 rpm) at 28°C for a period of 144 hrs. Later the biomass was harvested by centrifugation (6000 rpm) washed with distilled water twice and taken for extraction of carotenoids. The total carotenoid yield is 1250 µg/g dcm. EXAMPLE 17 For Enebose medium, the following ingredients, glucose (4.0g), yeast extract (0.15 g), CaCl2.H2O (0.04 g), MgSO4.7H20 (0.04 g), KH2PO4 (0.075 g) and NH4NO3 (0.0286 g) were weighed into a beaker, to which 90 ml of water was added. The ingredients are dissolved in water, pH adjusted to 5.5 with 0.1N NaOH/O.lN HC1. The final volume was made up to 92 ml and sterilised at 121°C for 20 min, cooled to 28+2°C. The sterilised extract with ingredients was inoculated with 10% of 20 hr old cell suspension of R.gracilis under sterile condition. This was incubated on a rotary shaker (200 rpm) at 28°C for a period of 120 hrs. Later the biomass is harvested by centrifugation (6000 rpm) washed with water twice and taken for extraction of carotenoids. The total carotenoid yield is 427 µg/g dcm.. EXAMPLE 18 Inoculum of R.gracilis was prepared by transferring a loop of 3 days old culture of R.gracilis from PDA slants into Erlenmeyer flask containing Enebose medium with 1% glucose. This was incubated on a rotary shaker at 200 rpm for 20 hr at 28+2°C which served as the initial inoculum. EXAMPLE 19 R.gracilis was cultivated in growth medium as follows: 10 ml of 20 hr old inoculum as described in Example 3 was transferred into 500 ml Erlenmeyer flasks containing 92 ml of growth mediums, the (Examples 1 to 9). Incubated on a rotary shaker (200 rpm) at a temperature of 28±2°C for 120 hr. At the end of fermentation, yeast was harvested by centrifugation (6000 rpm) for 15 minutes, washed twice with distilled water. The supernatant is discarded and the biomass was used for extraction of carotenoids. The main advantages of the present invention are: 1. Use of natural and low cost substrate 2. Low cost of production We Claim: 1. A process for the production of carotenoids from microbial source using wheat bran extract, which comprises: a) soaking, extracting followed by filtering of the wheat bran with water, at 20 - 97°C temperature, b) enriching of the above filtrate with carbon source such as herein described at 1.5 to 4.0% level and pH of 4.5 to 6.0m c) sterilizing the enriched extract at 115 - 125°C for 15 to 25 minutes followed by cooling to 27 to 32°C, d) inoculating the above sterilized extract with 8 to 12% yeast cell suspension as defined herein , e) fermenting aerobically for 36 to 168 hr in a state of rotary motion at 25 - 32°C, f) harvesting the above cell biomass from fermented broth for extraction of carotenoid by using an organic solvents. 2. A process as claimed in claims 1-2 wherein the extraction of wheat bran is carried out with water preferably at 20 - 35°C temperature. 3. A process as claimed in claims 1 - 3, wherein the carbon source used for enrichment is glucose preferably at 1 - 3% level maximum and pH 4.5 to 6.0. 4. A process as claimed in claims 1-4, wherein, the sterilization is carried out preferably at a temperature of 120 - 122°C for 18-22 minutes maximum. 5. A process as claimed in claims 1-5 wherein the yeast cell suspension is Rhodotorula gracilis at 8 to 12% concentration. 6. A process as claimed in claims 1 to 6 above, wherein the cell suspension is 16 - 24hrold. 7. A process as claimed in claims 1 - 7 wherein the broth is fermented aerobically for a period of 72-144 hr. 8. A process as claimed in claims 1-8 wherein the organic solvents used for extraction is selected from acetone, hexane and diethyl ether. 9. A process as claimed in claims 1 - 9 wherein the yield of total carotenoid is 1 100 to 1300µg/g dry cell mass. 10. A process for the production of carotenoid from microbial source using wheat bran extract substantially as herein described with reference to the examples.

Full Text

The present invention relates to a process for the production of carotenoid from microbial source.
Carotenoids are required as feed supplements in the poultry industry and in aquaculture of fishes and crustaceans. Besides, providing nutrition and possibly disease resistance, carotenoids give brilliant pigmentation and aesthetic value to Crustacea, animals, etc.
Recent studies have suggested that carotenoids have health benefits in humans and animals by preventing or delaying some chronic diseases including cancer, arteriosclerosis and cataract. These findings have contributed greatly to the industrial and medical interests in carotenoids.
Most of the carotenoids used in the food, feed, nutritional supplement, cosmetic, and pharmaceutical industries or for other industrial purposes are produced by total chemical synthesis. Production of natural food colours is gaining importance throughout the world, due to increase in public awareness and also due to the growing consumer demand for safe, natural food colourants. Because of technological limitations of synthetic colourants, there is a growing interest in the development and application of colourants from microbial sources. Certainly, a primary contributing factor to the increased interest in natural carotenoids is the current trend of avoiding food additives and synthetic chemicals in foods.
Microbial carotenoids have attracted much interest in recent years for several reasons, the primary reason is the growth of aquaculture - currently the fastest growing sector of agriculture. Astaxanthin is an important source of pigmentation, p-carotene, on the other hand, is an important food colourant as well as the source of vitamin-A in animal diets. The market for these two carotenoids is rapidly growing and only microbial synthesis can meet the demand for biological carotenoids.
The rapid growth of microbes reduces the production time to a matter of days and the process lends itself to a continuous operation.
Carotenoid pigment biosynthesis is a characteristic ability of genus Rhodotorula. It produces a mixture of carotenoids such as P-carotene, torulene and torularhodine. The P-carotene found in this yeast has attracted commercial interest as a precursor of vitamin-A and as a yellow pigment for food.
Reference may be made to the work carried out by Frengova et al. (1994) wherein whey filtrate was used as a substrate for formation of carotenoids by R.gracilis with co-cultivation of homo-fermentative lactic acid bacteria. The draw back in this method is a synthetic medium consisting of high concentration of glucose, yeast extract, phosphate and magnesium sulphate as growth medium which is expensive and also separation of carotenoids from a mixed culture is not suitable.
Reference may be made to Martin et al. (1993) wherein acid extracts of peat was used as the main substrate source for carotenoids production by Rhodotorula rubra. The draw back of this method is the supplementation of nutrients such as (NH4)2S04, yeast extract, K2SO4 and MgSO4.7H2O. Carotenoid concentration reported is 1,256 ug/g dry mass. Peat is not a commonly available substrate for use.
Reference may be made to Buzzini and Martini (1999) wherein grape must, glucose syrup, beet molasses, soyabean flour extract maize flour extract, etc were used individually as carbon substrate for carotenoid production. In these media, other nutrients such as glucose, KH2P04, MgS04.7H2O, yeast extract were also used. The total carotenoid reported with (a) concentrated grape must is 915.4 µg/g dry cells, (b) grape must 455.4 µg/g, (c) glucose syrup 240 µg/g, (d) beet (e) molasses 332.8 µg/g, (e) soyabean flour extract 883.3µg/g and with maize flour extract 383.9 µg/g dry cells only.
Reference may be made to the work carried out by Costa et al. (1987), wherein a chemical defined medium consisting of sucrose, (NH4)2SO4, KH2PO4, Na2HPO4, MgSO4.7H2O yeast extract, with pH 6.5 was used and 605 ug/g of dry cells of P-carotene is obtained when cells are maintained under non-proliferating conditions. This process involves an additional step of harvesting, washing and second stage of fermentation.
Reference may be made to the work carried out by Shih and Hang (1996), wherein sauerkraut brine is used as a substrate for production of carotenoids and the maximum yield of total carotenoids expressed as P-carotene was only 16.35 ug/g yeast on dry weight basis.
The technical know how of using wheat bran extract for carotenoid production under submerged fermentation is not established so far.
The main object of the present invention is a process for the production of carotenoid from microbial
source.
Accordingly, present invention provides a process for the production of carotenoids from microbial source
using wheat bran extract, which comprises:
a) soaking, extracting followed by filtering of the wheat bran with water, at 20 - 97°C temperature,
b) enriching of the above filtrate with carbon source such as herein described at 1.5 to 4.0% level and
pH of 4.5 to 6.Om
c) sterilizing the enriched extract at 115 - 125°C for 15 to 25 minutes followed by cooling to 27 to
32°C,
d) inoculating the above sterilized extract with 8 to 12% yeast cell suspension such as defined herein ,
e) fermenting aerobically for 36 to 168 hr in a state of rotary motion at 25 - 32°C,
f) harvesting the above cell biomass from fermented broth for extraction of carotenoid by using an
organic solvents.
In embodiment of the present invention extraction may be carried out with water at 20 to 97°C
temperature.
In another embodiment of the present invention, the carbon source used for enrichment may be glucose
at 1.5 to 4% maximum at pH 4.5 to 6.0.
In an embodiment of the present invention, the sterilization may be carried out at 120 - 122°C for 20 to 22
minutes.
In an another embodiment, the yeast cell suspension may contain Rhodotorula gracilis at 8 - 12 %
concentration.
Rhodotorula gracilus (ATCC 90950) produces a mixture of carotenoids such as torulene, torulahodine
and beta-carotene which find its application as food colourant in cosmetics, Pharmaceuticals and also in
poultry feed. The yeast in use produces more of beta-carotene. In addition, the yeast also produces
lipids which may add value to the product.
In another embodiment of the present invention, the cell suspension of Rhodotorula gracilis may be 20 to
24 hr old culture.
In an another embodiment, the broth may be fermented aerobically for a period of 36 - 168 hr.
In an another embodiment of the invention, the solvents used for extraction may be from acetone, hexane and diethyl ether.
In an embodiment of the invention the yield of total carotenoid may be 1100 to 1300 µg/g dry cell mass.
The detailed process for the production of carotenoid is presented below along with the flow diagram.
The detail process for production of carotenoid is as follows: R.gracilis CFR1 was maintained on PDA agar (slants) consisting per litre glucose 20.Og, potato infusion from 200g of potato, agar 20.0g at 4°C.
Preparation of wheat bran extract medium
l.Og of wheat bran was taken in a beaker, suspended in 50 ml of distilled water, left at ambient temperature for a period of one hour. After one hour of soaking, the wheat bran extract was filtered through a muslin cloth and centrifuged. The supernatant was collected in a conical flask. The residue was washed 3 times with water. The supernatant are pooled together and the residue discarded.
To the filtrate 2 g of glucose was added. The volume was made up to 85 ml. pH was adjusted to 5.5 with 0.1N NaOH/O.lN HC1. The final volume was made upto 92 ml. The medium was sterilised at 121°C for 20 min, cooled and used as medium for production of carotenoids. Preparation of inoculum
1% Enebose medium was used as growth medium for inoculum preparation. The following ingredients glucose 1 g, CaCl2.2H2O (0.04g), MgSO4.7H2O (0.04g), yeast extract (0.15 g), NH4N03 (0.0286 g), KH2PO4 (0.075 g) were weighed in a beaker, dissolved completely in 95 ml of water. pH was adjusted to 5.5 with 0.1N NaOH/O.lN HC1. The final volume was made up to 100 ml. This medium was taken in a 500 ml Erlenmeyer flasks, sterilised at 121°C for 20 min, cooled to room temperature. A loop of 3 days old culture of R.gracilis from PDA slants was transferred into Enebose medium with 1% glucose, incubated on a rotary shaker (200 rpm) at ambient temperature (28+2°C) for 20 hr. At the end of 20 hr, 10 ml of this cell suspension (fermented broth) was used for inoculation of wheat bran medium for carotenoid production.

Cultivation of yeast cells for carotenoid production
10 ml of 20 hr old cell culture of R.gracilis (107/ml) was inoculated into wheat bran medium (90 ml). This was incubated on a rotary shaker (200 rpm) at ambient temperature 28+2°C for 120 hours. Harvesting
At the end of fermentation, yeast cells were harvested by centrifugation (6000 rpm) for 15 min. The supernatant was discarded. The cells were washed twice with distilled water to remove excessive nutrients. This biomass was used for extraction of carotenoids.
Extraction of carotenoids was carried out as follows: 1.0 g of wet cells are taken in a pestle and mortar. Two spatula (10 g) full 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 a cylinder. Another 20 ml of acetone was added to the cells, ground to 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 solvent extracts are pooled together passed through a bed of anhydrous sodium sulphate. This clear carotenoid extract was flash evaporated at 35°C with 120 rpm under 200 Ib vacuum to dryness. The extract is taken in 10 ml of chloroform for estimation. Estimation of carotenoids
The carotenoid content were estimated by absorption spectrum of the carotenoid solution at 460 nm. A molar extinction coefficient value of 99330 cm/l/mol obtained from an average value of E from ten carotenoids (lycopene, alpha-carotene, ß-carotene, δ-carotene, zeaxanthin, rhodoxanthin, astaxanthin, lutein, ß-apo-2 carotenal and dihydro ß-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 measurements were carried out using a UV-160 recording Spectrophotometer (Shimadzu, Japan).
In the above process, other nutrients such as yeast extract, MgSO4. 7H2O, KH2PO4, NH4NO3 and CaCl2.H2O are eliminated. All these are replaced by just the water extract of wheat bran which was later enriched with glucose for the growth of the yeast for carotenoid production.
Flow diagram for the process
Wheat bran
Water retraction
Extract + Wg of glucose
pH adjusted to 5.5
Sterilised using autoclave Inoculated with N.gracilis cells 10%
Fermentation for 120hr at room temperature Harvest cellusing centrifuge Cells washedwith plain water
Extracted with solvents (hexane, acetone, diethyl ether)

imnatic
Concentrate and estimation of carotenoids
Novelty of the process
1. Use of wheat bran as substrate
2. Higher yield of carotenoid compared to any reported literature
3. Use of microbial source
EXAMPLE 1
0.5 g of wheat bran was taken in a beaker and was suspended in 50 ml of distilled water, left at 20°C for a period of one hour. After one hour of soaking, the mixture (wheat bran + water) was filtered through a muslin cloth and then centrifuged. The centrifuged filtrate was collected in a conical flask. The residue was washed 3 times with 10 ml of distilled water each. The filtrates are pooled together and the residue was discarded.
To the filtrate, the following ingredients: glucose (4.0g), CaCl2.H2O (0.04 g), MgSO4.7H2O (0.04 g), KH2PO4 (0.075 g) and NH4NO3 (0.0286 g) were added, dissolved completely by mixing. The volume was made up to 85 ml, pH was adjusted to 5.5 with 0.1N NaOH/O.lN HC1. The final volume was made upto 92 ml. The medium was sterilised at 121°C for 20 minutes, cooled to 28+2°C. The sterilised extract with ingredients was inoculated with 10% of 16 hr old cell suspension ofR.gracilis under sterile condition. This was incubated on a rotary shaker (200 rpm) at 25°C for a period of 144 hrs. Later the biomass is harvested by centrifugation (6000 rpm) washed with water twice and taken for extraction of carotenoids.
The total carotenoid yield is 510 µg/g dcm.
EXAMPLE 2
1.0 g of wheat bran was taken in a beaker and was suspended in 50 ml of distilled water, left at 28°C for a period of one hour. After one hour of soaking, the mixture (whet bran + water) was filtered through a muslin cloth and centrifuged. The centrifuged filtrate was collected in a conical flask. The residue was washed 3 times with 10 ml of distilled water. The filtrates are pooled together and the residue was discarded.
To the filtrate, the following ingredients: glucose (4.0g), CaCl2.H2O (0.04 g), MgSO4.7H2O (0.04 g), KH2PO4 (0.075 g) and NH4NO3 (0.0286 g) were added, dissolved completely by mixing. The volume was made up to 85 ml, pH was adjusted to 5.5 with 0.1N NaOH/O.lN HC1. The final volume made upto 92
ml. The medium was sterilised at 121°C for 20 minutes, cooled to 28±2°C. The sterilised extract with ingredients was inoculated with 10% of 20 hr old cell suspension ofR.gracilis under sterile condition. This was incubated on a rotary shaker (200 rpm) at 28°C for a period of 144 hrs. Later the biomass is harvested by centrifugation (6000 rpm) washed with water twice and taken for extraction of carotenoids. The total carotenoid yield is 630µg/g dcm.
EXAMPLES
2.0 g of wheat bran was taken in a beaker and was suspended in 50 ml of distilled water, left at 32°C for one hour. After one hour of soaking, the mixture (whet bran + water) was filtered through a muslin cloth and centrifuged. This centrifuged filtrate was collected in a conical flask. The residue was washed 3 times with 10 ml of distilled water. The filtrates are pooled together and the residue was discarded.
To the filtrate, the following ingredients: glucose (4.0g), CaCl2.H2O (0.04 g), MgSO4.7H2O (0.04 g), KH2P04 (0.075 g) and NH4NO3 (0.0286 g) were added, dissolved completely by mixing. The volume was made up to 85 ml, pH was adjusted to 5.5 with O.IN NaOH/O.lN HCI. The final volume made upto 92 ml. The medium was sterilised at 121°C for 20 minutes, cooled to 28+2°C. The sterilised extract with ingredients was inoculated with 10% of 16 hr old cell suspension ofR.gracilis under sterile condition. This was incubated on a rotary shaker (200 rpm) at 32°C for a period of 144 hrs. Later the biomass is harvested by centrifugation (6000 rpm) washed with water twice and taken for extraction of carotenoids. The total carotenoid yield is 410 jig/g dcm.
EXAMPLE 4
0.5 g of wheat bran was taken in a beaker. 60 ml of distilled water was added and boiled (97°C) on a slow flame for 15 minutes, cooled and filtered through a muslin cloth and centrifuged. The centrifuged filtrate was collected in a conical flask. The cooled residue was further washed 2 times with 10 ml of water. All the filtrates were pooled together and the residue was discarded.
To the filtrate, the following ingredients: glucose (4.0g), CaCl2.H2O (0.04 g), MgSO4.7H20 (0.04 g), KH2P04 (0.075 g) and NH4NO3 (0.0286 g) were added, dissolved completely by mixing. The volume
was made up to 85 ml, PH was adjusted to 5.5 with 0.1N NaOH/O.lN HC1. The final volume made upto 92 ml. The medium was sterilised at 121°C for 20 minutes, cooled to 28±2°C. The sterilised extract with ingredients was inoculated with 10% of 24 hr old cell suspension ofR.gracilis under sterile condition. This was incubated on a rotary shaker (200 rpm) at 28°C for a period of 120 hrs. Later the biomass is harvested by centrifugation (6000 rpm) washed with water twice and taken for extraction of carotenoids. The total carotenoid yield is 540 µg/g dcm.
EXAMPLES
1.0 g of wheat bran was taken in a beaker. 60 ml of distilled water was added and boiled (97°C) on a slow flame for 15 minutes, cooled and filtered through a muslin cloth and centrifuged. The centrifuged filtrate was collected in a conical flask. The cooled residue was further washed 2 times with 10 ml of water. All the filtrates were pooled together and the residue was discarded.
To the filtrate, the following ingredients: glucose (4.0g), CaCl2.H20 (0.04 g), MgSO4.7H2O (0.04 g), KH2PO4 (0.075 g) and NH4NO3 (0.0286 g) were added, dissolved completely by mixing. The volume was made up to 85 ml, pH was adjusted to 5.5 with 0.1N NaOH/O.lN HC1. The final volume made upto 92 ml. The medium was sterilised at 121°C for 20 minutes, cooled to 28±2°C. The sterilised extract with ingredients was inoculated with 10% of 20 hr old cell suspension ofR.gracilis under sterile condition. This was incubated on a rotary shaker (200 rpm) at 28°C for a period of 144 hrs. Later the biomass is harvested by centrifugation (6000 rpm) washed with water twice and taken for extraction of carotenoids. The total carotenoid yield is 522 ug/g dcm.
EXAMPLE 6
2.0 g of wheat bran was taken in a beaker. 60 ml of distilled water was added and boiled on a slow flame for 15 minutes (97°C), cooled and filtered through a muslin cloth and centrifuged. The centrifuged filtrate was collected in a conical flask. The cooled residue was further washed 2 times with 10 ml of water. All the filtrates were pooled together and the residue was discarded.
To the filtrate, the following ingredients: glucose (4.0g), CaCl2.H2O (0.04 g), MgSO4.7H2O (0.04 g), KH2PO4 (0.075 g) and NH4NO3 (0.0286 g) were added, dissolved completely by mixing. The volume
was made up to 85 ml, pH was adjusted to 5.5 with 0.1N NaOH/O.lN HC1. The final volume made upto 92 ml. The medium was sterilised at 121°C for 20 minutes, cooled to 28±2°C. The sterilised extract with ingredients was inoculated with 8% of 20 hr old cell suspension of R.gracilis under sterile condition. This was incubated on a rotary shaker (200 rpm) at 28°C for a period of 120 hrs. Later the biomass is harvested by centrifugation (6000 rpm) washed with water twice and taken for extraction of carotenoids. The total carotenoid yield is 470 µg/g dcm.
EXAMPLE 7
1.0 g of wheat bran was taken in a beaker, and suspended in 50 ml of distilled water. Left at 28°C for a period of one hour. After one hour of soaking, the mixture (wheat bran + water) was filtered through a muslin cloth and then centrifuged. The centrifuged filtrate was collected in a conical flask. The residue was washed 3 times with 10 ml of distilled water. The filtrates were pooled together and the residue was discarded. To the filtrate, 1.5 g of glucose was added, dissolved completely by mixing. The volume is made upto 85 ml, pH was adjusted to 5.5 with 0.1N NaOH/O.lN HC1. The final volume was made upto 92 ml. The medium was sterilised at 121°C for 20 minutes and cooled. The sterilised extract with glucose was inoculated with 10% of 20 hr old culture suspension of R.gracilis under sterile condition. This was incubated on a rotary shaker (200 rpm) at 28°C for a period of 120 hrs. Later the biomass was harvested by centrifugation (6000 rpm) washed with distilled water twice and taken for extraction of carotenoids. The total carotenoid yield is 1100 µg/g dcm.
EXAMPLE 8
1.0 g of wheat bran was taken in a beaker, and suspended in 50 ml of distilled water. Left at 28°C for a period of one hour. After one hour of soaking, the mixture (wheat bran + water) was filtered through a muslin cloth and then centrifuged. The centrifuged filtrate was collected in a conical flask. The residue was washed 3 times with 10 ml of distilled water. The filtrates were pooled together and the residue was discarded. To the filtrate, 3 g of glucose was added, dissolved completely by mixing. The volume is made upto 85 ml, pH was adjusted to 5.5 with 0.1N NaOH/O.lN HC1. The final volume was made upto 92 ml. The medium was sterilised at 121°C for 20 minutes and cooled. The sterilised extract with glucose was
inoculated with 10% of 20 hr old culture suspension of R.gracilis under sterile condition. This was incubated on a rotary shaker (200 rpm) at 28°C for a period of 120 hrs. Later the biomass was harvested by centrifugation (6000 rpm) washed with distilled water twice and taken for extraction of carotenoids. The total carotenoid yield is 800 µg/g dcm.
EXAMPLE 9
1.0 g of wheat bran was taken in a beaker, and suspended in 50 ml of distilled water. Left at 28°C for a period of one hour. After one hour of soaking, the mixture (wheat bran + water) was filtered through a muslin cloth and then centrifuged. The centrifuged filtrate was collected in a conical flask. The residue was washed 3 times with 10 ml of distilled water. The filtrates were pooled together and the residue was discarded. To the filtrate, 4 g of glucose was added, dissolved completely by mixing. The volume is made upto 85 ml, pH was adjusted to 5.5 with 0.1N NaOH/O.lN HC1. The final volume was made upto 92 ml. The medium was sterilised at 121°C for 20 minutes and cooled. The sterilised extract with glucose was inoculated with 10% of 20 hr old culture suspension of R.gracilis under sterile condition. This was incubated on a rotary shaker (200 rpm) at 28°C for a period of 120 hrs. Later the biomass was harvested by centrifugation (6000 rpm) washed with distilled water twice and taken for extraction of carotenoids. The total carotenoid yield is 900µg/g dcm.
EXAMPLE 10
1.0 g of wheat bran was taken in a beaker, and suspended in 50 ml of distilled water. Left at 28°C for a period of one hour. After one hour of soaking, the mixture (wheat bran + water) was filtered through a muslin cloth and then centrifuged. The centrifuged filtrate was collected in a conical flask. The residue was washed 3 times with 10 ml of distilled water. The filtrates were pooled together and the residue was discarded. To the filtrate, 2 g of glucose was added, dissolved completely by mixing. The volume is made
upto 85 ml, pH was adjusted to 4.5 with O.IN NaOH/O.lN HCI. The final volume was made upto 92 ml. The medium was sterilised at 121°C for 20 minutes and cooled. The sterilised extract with glucose was inoculated with 10% of 20 hr old culture suspension of R.gracilis under sterile condition. This was incubated on a rotary shaker (200 rpm) at 28°C for a period of 120 hrs. Later the biomass was harvested by centrifugation (6000 rpm) washed with distilled water twice and taken for extraction of carotenoids. The total carotenoid yield is 1000 µg/g dcm.
EXAMPLE 11
1.0 g of wheat bran was taken in a beaker, and suspended in 50 ml of distilled water. Left at 28°C for a period of one hour. After one hour of soaking, the mixture (wheat bran + water) was filtered through a muslin cloth and then centrifuged. The centrifuged filtrate was collected in a conical flask. The residue was washed 3 times with 10 ml of distilled water. The filtrates were pooled together and the residue was discarded. To the filtrate, 2.0 g of glucose was added, dissolved completely by mixing. The volume is made upto 85 ml, pH was adjusted to 5.5 with O.IN NaOH/O.lN HCI. The final volume was made upto 92 ml. The medium was sterilised at 121°C for 20 minutes and cooled. The sterilised extract with glucose was inoculated with 10% of 20 hr old culture suspension of R.gracilis under sterile condition. This was incubated on a rotary shaker (200 rpm) at 28°C for a period of 120 hrs. Later the biomass was harvested by centrifugation (6000 rpm) washed with distilled water twice and taken for extraction of carotenoids.
The total carotenoid yield is 1300 µg/g dcm.
EXAMPLE 12
1.0 g of wheat bran was taken in a beaker, and suspended in 50 ml of distilled water. Left at 28°C for a period of one hour. After one hour of soaking, the mixture (wheat bran + water) was filtered through a muslin cloth and then centrifuged. The centrifuged filtrate was collected in a conical flask. The residue was washed 3 times with 10 ml of distilled water. The filtrates were pooled together and the residue was discarded. To the filtrate, 2.0 g of glucose was added, dissolved completely by mixing. The volume is
made upto 85 ml, pH was adjusted to 6.0 with 0.1N NaOH/O.lN HCI. The final volume was made upto 92 ml. The medium was sterilised at 121°C for 20 minutes and cooled. The sterilised extract with glucose was inoculated with 10% of 20 hr old culture suspension of R.gracilis under sterile condition. This was incubated on a rotary shaker (200 rpm) at 28°C for a period of 168 hrs. Later the biomass was harvested by centrifugation (6000 rpm) washed with distilled water twice and taken for extraction of carotenoids. The total carotenoid yield is 720 µg/g dcm.
EXAMPLE 13
1.0 g of wheat bran was taken in a beaker, and suspended in 50 ml of distilled water. Left at 28°C for a period of one hour. After one hour of soaking, the mixture (wheat bran + water) was filtered through a muslin cloth and then centrifuged. The centrifuged filtrate was collected in a conical flask. The residue was washed 3 times with 10 ml of distilled water. The filtrates were pooled together and the residue was discarded. To the filtrate, 1.5 g of glucose was added, dissolved completely by mixing. The volume is made upto 85 ml, pH was adjusted to 5.5 with 0. IN NaOH/0. IN HCI. The final volume was made upto 92 ml. The medium was sterilised at 121°C for 20 minutes and cooled. The sterilised extract with glucose was inoculated with 6% of 20 hr old culture suspension of R.gracilis under sterile condition. This was incubated on a rotary shaker (200 rpm) at 28°C for a period of 120 hrs. Later the biomass was harvested by centrifugation (6000 rpm) washed with distilled water twice and taken for extraction of carotenoids.
The total carotenoid yield is 937 µg/g dcm.
EXAMPLE 14
1.0 g of wheat bran was taken in a beaker, and suspended in 50 ml of distilled water. Left at 28°C for a period of one hour. After one hour of soaking, the mixture (wheat bran + water) was filtered through a muslin cloth and then centrifuged. The centrifuged filtrate was collected in a conical flask. The residue was washed 3 times with 10 ml of distilled water. The filtrates were pooled together and the residue was

discarded. To the filtrate, 2.0 g of glucose was added, dissolved completely by mixing. The volume is made upto 85 ml, PH was adjusted to 5.5 with O.IN NaOH/O.lN HCI. The final volume was made upto 92 ml. The medium was sterilised at 121°C for 20 minutes and cooled. The sterilised extract with glucose was inoculated with 12% of 20 hr old culture suspension of R.gracilis under sterile condition. This was incubated on a rotary shaker (200 rpm) at 28°C for a period of 120 hrs. Later the biomass was harvested by centrifugation (6000 rpm) washed with distilled water twice and taken for extraction of carotenoids. The total carotenoid yield is 1170 µg/g dcm.
EXAMPLE 15
1.0 g of wheat bran was taken in a beaker, and suspended in 50 ml of distilled water. Left at 28°C for a period of one hour. After one hour of soaking, the mixture (wheat bran + water) was filtered through a muslin cloth and then centrifuged. The centrifuged filtrate was collected in a conical flask. The residue was washed 3 times with 10 ml of distilled water. The filtrates were pooled together and the residue was discarded. To the filtrate, 2.0 g of glucose was added, dissolved completely by mixing. The volume is made upto 85 ml, pH was adjusted to 5.5 with O.IN NaOH/O.lN HCI. The final volume was made upto 92 ml. The medium was sterilised at 121°C for 20 minutes and cooled. The sterilised extract with glucose was inoculated with 10% of 20 hr old culture suspension of R.gracilis under sterile condition. This was incubated on a rotary shaker (200 rpm) at 28°C for a period of 36 hrs. Later the biomass was harvested by centrifugation (6000 rpm) washed with distilled water twice and taken for extraction of carotenoids.
The total carotenoid yield is 43 µg/g dcm.
EXAMPLE 16
1.0 g of wheat bran was taken in a beaker, and suspended in 50 ml of distilled water. Left at 28°C for a period of one hour. After one hour of soaking, the mixture (wheat bran + water) was filtered through a muslin cloth and then centrifuged. The centrifuged filtrate was collected in a conical flask. The residue was washed 3 times with 10 ml of distilled water. The filtrates were pooled together and the residue was
discarded. To the filtrate, 2.0 g of glucose was added, dissolved completely by mixing. The volume is made upto 85 ml, pH was adjusted to 5.5 with 0.1N NaOH/O.lN HC1. The final volume was made upto 92 ml. The medium was sterilised at 121°C for 20 minutes and cooled. The sterilised extract with glucose was inoculated with 10% of 20 hr old culture suspension of R.gracilis under sterile condition. This was incubated on a rotary shaker (200 rpm) at 28°C for a period of 144 hrs. Later the biomass was harvested by centrifugation (6000 rpm) washed with distilled water twice and taken for extraction of carotenoids. The total carotenoid yield is 1250 µg/g dcm.
EXAMPLE 17
For Enebose medium, the following ingredients, glucose (4.0g), yeast extract (0.15 g), CaCl2.H2O (0.04 g), MgSO4.7H20 (0.04 g), KH2PO4 (0.075 g) and NH4NO3 (0.0286 g) were weighed into a beaker, to which 90 ml of water was added. The ingredients are dissolved in water, pH adjusted to 5.5 with 0.1N NaOH/O.lN HC1. The final volume was made up to 92 ml and sterilised at 121°C for 20 min, cooled to 28+2°C. The sterilised extract with ingredients was inoculated with 10% of 20 hr old cell suspension of R.gracilis under sterile condition. This was incubated on a rotary shaker (200 rpm) at 28°C for a period of 120 hrs. Later the biomass is harvested by centrifugation (6000 rpm) washed with water twice and taken for extraction of carotenoids.
The total carotenoid yield is 427 µg/g dcm..
EXAMPLE 18
Inoculum of R.gracilis was prepared by transferring a loop of 3 days old culture of R.gracilis from PDA slants into Erlenmeyer flask containing Enebose medium with 1% glucose. This was incubated on a rotary shaker at 200 rpm for 20 hr at 28+2°C which served as the initial inoculum.
EXAMPLE 19
R.gracilis was cultivated in growth medium as follows: 10 ml of 20 hr old inoculum as described in Example 3 was transferred into 500 ml Erlenmeyer flasks containing 92 ml of growth mediums, the
(Examples 1 to 9). Incubated on a rotary shaker (200 rpm) at a temperature of 28±2°C for 120 hr. At the end of fermentation, yeast was harvested by centrifugation (6000 rpm) for 15 minutes, washed twice with distilled water. The supernatant is discarded and the biomass was used for extraction of carotenoids.
The main advantages of the present invention are:
1. Use of natural and low cost substrate
2. Low cost of production

We Claim:
1. A process for the production of carotenoids from microbial source using wheat
bran extract, which comprises:
a) soaking, extracting followed by filtering of the wheat bran with water, at 20 -
97°C temperature,
b) enriching of the above filtrate with carbon source such as herein described at 1.5
to 4.0% level and pH of 4.5 to 6.0m
c) sterilizing the enriched extract at 115 - 125°C for 15 to 25 minutes followed by
cooling to 27 to 32°C,
d) inoculating the above sterilized extract with 8 to 12% yeast cell suspension as
defined herein ,

e) fermenting aerobically for 36 to 168 hr in a state of rotary motion at 25 - 32°C,
f) harvesting the above cell biomass from fermented broth for extraction of
carotenoid by using an organic solvents.

2. A process as claimed in claims 1-2 wherein the extraction of wheat bran is
carried out with water preferably at 20 - 35°C temperature.
3. A process as claimed in claims 1 - 3, wherein the carbon source used for
enrichment is glucose preferably at 1 - 3% level maximum and pH 4.5 to 6.0.
4. A process as claimed in claims 1-4, wherein, the sterilization is carried out
preferably at a temperature of 120 - 122°C for 18-22 minutes maximum.
5. A process as claimed in claims 1-5 wherein the yeast cell suspension is
Rhodotorula gracilis at 8 to 12% concentration.
6. A process as claimed in claims 1 to 6 above, wherein the cell suspension is 16 -
24hrold.

7. A process as claimed in claims 1 - 7 wherein the broth is fermented aerobically
for a period of 72-144 hr.
8. A process as claimed in claims 1-8 wherein the organic solvents used for
extraction is selected from acetone, hexane and diethyl ether.
9. A process as claimed in claims 1 - 9 wherein the yield of total carotenoid is 1 100
to 1300µg/g dry cell mass.
10. A process for the production of carotenoid from microbial source using wheat
bran extract substantially as herein described with reference to the examples.

Documents:

382-del-2001-abstract.pdf

382-del-2001-claims.pdf

382-del-2001-correspondence-others.pdf

382-del-2001-correspondence-po.pdf

382-del-2001-description (complete).pdf

382-del-2001-form-1.pdf

382-del-2001-form-18.pdf

382-del-2001-form-2.pdf

382-del-2001-form-3.pdf

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Patent Number

232500

Indian Patent Application Number

382/DEL/2001

PG Journal Number

13/2009

Publication Date

27-Mar-2009

Grant Date

17-Mar-2009

Date of Filing

29-Mar-2001

Name of Patentee

COUNCIL OF SCIENTIFIC & INDUSTRIAL RESEARCH

Applicant Address

RAFI MARG, NEW DELHI-110001, INDIA.

Inventors:

#	Inventor's Name	Inventor's Address
1	GOVINDASWAMY VIJAYALAKSHMI	FOOD MICROBIOLOGY DEPARTMENT CENTRAL FOOD TECHNOLOGICAL RESEARCH INSTITUTE, MYSORE-570013, INDIA.
2	VASUDEVA VANAJAHSHI	FOOD MICROBIOLOGY DEPARTMENT CENTRAL FOOD TECHNOLOGICAL RESEARCH INSTITUTE, MYSORE-570013, INDIA.

PCT International Classification Number

C12P 23/00

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA