Indian Patents. 215665:A PROCESS FOR THE PRODUCTION OF A NEW GROWTH MEDIUM FORMULATION FOR ENHANCED PRODUCTION OF CAROTENOIDS.

Title of Invention	A PROCESS FOR THE PRODUCTION OF A NEW GROWTH MEDIUM FORMULATION FOR ENHANCED PRODUCTION OF CAROTENOIDS.
Abstract	New growth medium formulation for enhanced production of carotenoids which comprises conventional assemblies carbon source (g/l) 37.5-42.5; MgSO47H2O 3.75-4.25; CaCI2 3.75-4.25; KH2P04 0.6-0.8; yeast extract 1.25-1.75; NH4N03 4.25-4.65 & pH 5.0-6.0 to obtain new growth medium formulation for enhanced production of carotenoids

Full Text	The present invention relates to new growth medium formulation for enhanced production of carotenoids. This invention particularly relates to a growth medium useful for enhanced production of carotenoid by Rhodotorula gracilis. There has been much interest in the development of new natural colorants for use in food industry. Microbes have recently been recognised as an important source of natural pigments. Rhodotorula, a red yeast produces carotenoid pigments which have great potential for application in food and feed industry both as a colourant and antioxidant in food formulations. The major carotenoid pigments produced by Rhodotorula are p-carotene, torulene and torularhodin. In the present patent, we disclose an innovative formulation of growth medium which effects increase in carotenoid production in comparison with yields in traditional media for yeasts. There are only a few reports on the carotenoids of Rhodotorula yeasts. Reference may be made to Simpson et al., (1964) who were the first to analyse the various pigments produced by Rhodotorula yeasts and also to propose a biosynthetic pathway for carotenoids (Simpson et al. 1964, J.Bact. 88, p-1688- 1694). The media used by these authors contains 10 % yeast autolysate and 5% glucose (pH 6.0). Reference may be made to Costa et al., (1989) wherein they have studied the production of carotenoids by Rhodotorula strain and reported to produce 630µg/g of dried cells and the media composition are as follows: (g/1) sucrose 20.0; (NH4)2SO4 3.7; KH2PO4 5.5; Na2HPO4 3.74; MgSO4 7H2O 0.5, yeast extract 1.0, pH 6.5. (Costa et al. 1989, Biotech. Lett., 9, 373-375). The production of carotenoids was noticed in the stationary phase and in non-proliferating conditions. Reference may also be made to Matelli et al., (1993) wherein they have reported the production of carotenoids by a Rhodotorula strain grown in sugarcane juice containing (g/1) (NH4)2SO4 5.3; KH2PO4 5.5; Na2HPO4 3.7; MgSO4 7H2O 0.5, yeast extract 1.0, pH 6.5 (Matelli et al. J.Fer. Bioeng. 1993). They have found that under non-proliferating conditions the pigment production was 2500 µg/g dried cells. Reference may also be made to Frengova et al., (1994) wherein a mixed culture of Rhodotorula glutinis and Lactobacillus in whey filtrate were able to produce 268 ug/g dry cells. The media compostion used for carotenoid production is whey filtrate medium containing (g/1) lactose 39.0, (NH4)2SO4 8.0; KH2PO4 3.0; MgSO4 7H2O 0.5, yeast extract 3.0, pH 5.3. Apart from these yeasts, the carotenoids are also produced in plants and many algae especially Dunaliella, which can accumulate 10 % of its cell dry weight as carotenoids. The drawbacks connected with hitherto known processes are as follows: In many of the processes using Rhodotomla yeasts the yields of carotenoids were low (268-2500 ug/g of yeast on dry wt basis). Although some of the plants and algal sources produce better yield of carotenoids than Rhodotomla, their cultivation pose many problems. They require sunlight and in some cases high salt concentrations for their growth (e.g., Dunaliella). The quality of carotenoids is not consistent as they are subjected to vagaries of nature. The production of carotenoid pigments canot be easily controlled like in many heterotrophic organisms. Carotenoids are gaining importance as natural food colorants in view of the possible safety and their synthetic counterparts being considered as harmful to health. Carotenoids function as antioxidants and some of them are precursors for vitamin A. The yield of carotenoids by Rhodotomla yeasts from hitherto known processes were low (268-2500 ug/g yeast on dry wt basis). The main object of the invention is to provide a process for the production of new growth medium formulation for the production of carotenoids in Rhodotomla which obviates the drawbacks as detailed above. Another object o the present invention is to check the influence of carbon/nitrogen ratio of media on the production of carotenoids. Accordingly the present invention provides a process for the production of a new growth medium formulation for enhanced production of carotenoids which comprises mixing of conventional assemblies carbon source (g/l) 37.5-42.5; MgSO47H2O 3.75-4.25; CaCI2 3.75-4.25; KH2P04 0.6-0.8; yeast extract 1.25-1.75; NH4N03 4.25-4.65 & pH 5.0-6.0 to obtain a new growth medium formulation for enhanced production of carotenoids. The C/N raito of the m3edium is 10 to 40 preferably 10. Rhodotorula gracilis was grown on this medium for 72 h and cells were harvested fro carotenoid estimation. The maximum total carotenoids production was at 72 h and it was in the range of 25740 to 25990 ug/g dry cells when C/N ratio is 10. In an embodiment of the present invention carotenoid production was found to occur later than lipid and low C/N ratio being more favourable than high C/N ratio for carotenoid production in Rh. Gracilies. In an another embodiment of the present invention the high carotenid production was seen in the medium having low pH. Hence the acidic pH of the medium was more suitable for the production of carotenoids. In yet another embodiment of the present invention the carotenoid. formation was not found to be associated with the cell growth and the maximum production was reached at the stationary phase. Rhodotorula gracilis CFR-1 (ATCC 90950) a locally isolated strain of red yeast was grown in different media as given in examples. The organism was grown in Erlenmayer flasks of 500 ml capacity having 250 ml of the above media. The flasks were incubated at 28±2°C on a rotary shaker. The biomass of the culture broth was determined by gravimetry. The cells contain approximately 70% moisture. 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 Rhodotorula gracilis was grown on Minimal salts medium-1 (MSM-1: glucose 40.0, MgSO47H2O 4.0, CaCl2 4.0, KH2PO4 0.75, yeast extract 1.5, NH4NO3 0.286 (g/1), pH 5.5) having a C/N ratio of 160. For comparison this yeast was also grown on the standard YEPD medium (yeast extract-peptone-dextrose (g/1)- yeast extract 10, peptone 20, glucose 20, pH 5.5) and Potato dextrose broth (PDB- (g/1) potato infusion from 200 g, glucose 20, pH 5.5). The carotenoids were estimated at different growth periods like 24, 48, 72, 96 and 120 h and the cells were harvested by centrifugation for 15 min at 5000 rpm. Cellbiomass was washed with distilled water. 2 g cells were taken in a pestle and mortar and ground with 8 g of sand (acid washed) in the acetone. The pigment was extracted with acetone till all the color was extracted from the cells (30 ml). The acetone extracts were centrifuged at 10,000 rpm for 20 min. Acetone extracts were transferred to 20 ml petroleum ether in a separating funnel and washed with 300 ml of distilled water . The petroleum ether phase was taken in a volumetric flask and the absorbance was read at 485 nm. Carotenoid production occurred very late in the stationary phase in YEPD medium and the total carotenoid production at 120 h was 1610 ug/g dry cells. EXAMPLE - 2 The organism was grown on Potato dextrose broth (PDB- (g/1) potato infusion from 200 g, glucose 20, pH 5.5) and the carotenoids were estimated at different growth periods like 24, 48, 72 h and the cells were harvested by centrifugation for 15 min at 5000 rpm. Cellbiomass was washed with distilled water. 2 g cells were taken in a pestle and mortar and ground with 8 g of sand (acid washed) in the acetone. The pigment was extracted with acetone till all the color was extracted from the cells (30 ml). The acetone extracts were centrifuged at 10,000 rpm for 20 min. Acetone extracts were transferred to 20 ml petroleum ether (60-80°C( in a separating funnel and washed with 300 ml of distilled water . The petroleum ether phase was taken in a volumetric flask and the absorbance was read at 485 nm. This medium with complex nitrogen source favoured carotenoid production at very early stationary phase (at 48 h). The total carotenoid production was maximum at 72 h and the yield was 2446 µg/g dry cells. EXAMPLE-3 The organism was grown on Minimal salts medium-1 (MSM-1: glucose 40.0, MgSO47H2O 4.0, CaCl2 4.0, KH2PO4 0.75, yeast extract 1.5, NH4NO3 0.286 (g/1), pH 5.5) having a C/N ratio of 160 and the carotenoids were estimated at different growth periods like 24, 48, 72, 96 h and the cells were harvested by centrifugation for 15 min at 5000 rpm. Cellbiomass was washed with distilled water. 2 g cells were taken in a pestle and mortar and ground with 8 g of sand (acid washed) in the acetone. The pigment was extracted with acetone till all the color was extracted from the cells (30 ml). The acetone extracts were centrifuged at 10,000 rpm for 20 min. Acetone extracts were transferred to 20 ml petroleum ether (60-80°C) in a separating funnel and washed with 300 ml of distilled water . The petroleum ether phase was taken in a volumetric flask and the absorbance was read at 485 nm. The carotenoid production starts at 72 h and maximum at 96 h (1796 ug/g dry cells. The potato dextrose medium with complex nitrogen source favoured carotenoid production at very early stationary phase (at 48 h). The total carotenoid production was maximum at 72 h and the yield was 2446 ug/g dry cells. The carotenoid production in Minimal salts medium-1 with C/N ratio 160 starts at 72 h and was maximum at 96 h (1716 ug/g dry cells). Table 1. Carotenoid production in different media (Table Removed) EXAMPLE - 4 Rhodotorula gracilis was grown on Minimal salts salts medium-2 (MSM-2 (g/1) - glucose 40.0, MgSO47H2O 4.0, CaCl2 4.0, KH2PO4 0.75, yeast extract 1.5, NH4NO3 1.144 pH 5.5) having a C/N ratio of 40. For comparison this yeast was also grown on standard YEPD medium (yeast extract-peptone-dextrose (g/1)- yeast extract 10, peptone 20, glucose 20, pH 5.5) and Potato dextrose broth (PDB- (g/1) potato infusion from 200 g , glucose 20, pH 5.5). The carotenoids were estimated at different growth periods like 24, 48, 72, 96 and 120 h and the cells were harvested by centrifugation for 15 min at 5000 rpm. Cellbiomass was washed with distilled water. 2 g cells were taken in a pestle and mortar and ground with 8 g of sand (acid washed) in the acetone. The pigment was extracted with acetone till all the color was extracted from the cells (30 ml). The acetone extracts were centrifuged at 10,000 rpm for 20 min. Acetone extracts were transferred to 20 ml petroleum ether in a separating funnel and washed with 300 ml of distilled water . The petroleum ether phase was taken in a volumetric flask and the absorbance was read at 485 nm. Carotenoid production occurred very late in the stationary phase in YEPD medium and the total carotenoid production at 120 h was 1610 ug/g dry cells. The potato dextrose medium with complex nitrogen source favoured carotenoid production at very early stationary phase (at 48 h). The total carotenoid production was maximum at 72 h and the yield was 2446 ug/g dry cells. The carotenoid production in Minimal salts medium -2 starts at 48 h and was maximum at 72 h (2653 ug/g dry cells). Table 2. Carotenoid production in different media (Table Removed) EXAMPLE -5 Rhodotorula gracilis was grown on Minimal salts medium -3 (MSM-3 comprises conventional assimibile carbon source (g/1) 40.0; MgSO47H2O 4.0; CaCl2 4.0; KH2PO4 0.75; yeast extract 1.50; NH4NO3 4.576, pH 5.5) having a C/N ratio of 10. For comparison this yeast was also grown on standard YEPD medium (yeast extract-peptone-dextrose (g/1)- yeast extract 10, peptone 20, glucose 20, pH 5.5) ; Potato dextrose broth (PDB- (g/1) potato infusion from 200 g , glucose 20, pH 5.5). The carotenoids were estimated at different growth periods like 24, 48, 72, 96 and 120 h and the cells were harvested by centrifugation for 15 min at 5000 rpm. Cellbiomass was washed with distilled water. 2 g cells were taken in a pestle and mortar and ground with 8 g of sand (acid washed) in the acetone. The pigment was extracted with acetone till all the color was extracted from the cells (30 ml). The acetone extracts were centrifuged at 10,000 rpm for 20 min. Acetone extracts were transferred to 20 ml petroleum ether in a separating funnel and washed with 300 ml of distilled water . The petroleum ether phase was taken in a volumetric flask and the absorbance was read at 485 nm. Carotenoid production occured very late in the stationary phase in YEPD meium and the total carotenoid production at 120 h was 1610 ug/g dry cells. The potato dextrose medium with complex nitrogen source favoured carotenoid production at very early stationary phase (at 48 h). The total carotenoid production was maximum at 72 h and the yield was 2446 ug/g dry cells. The organism produces maximum amount of carotenoids at 72 h in Minimal salts medium -3 and it was 25990 ug/g dry cells. Table 3. Carotenoid production in different media (Table Removed) The main advantages of the present invention are: 1. A new culture medium for enhanced production of carotenoids by Rhodotorula gracilis. 2. Medium formulation; Minimal salts medium-3 favours the maximum production of carotenoid in Rhodotorula gracilis which is more than 10 times higher than the value reported in literature for production of carotenoids by Rhodotorula. 3. The rapid growth of yeasts cuts production time to a matter of days and the process lends itself to continuous operation. 4. Compared to plants or animal sources, the present production process from Rhodotorula gracilis is flexible and can be controlled. The composition/formulation obtained by the process of the present invention is neither a product of chemical reaction nor a mere admixture but is a synergistic formulation having properties different than the aggregate properties of the individual components We Claim: 1. A process for the production of a new growth medium formulation for enhanced production of carotenoids which comprises mixing of conventional assemblies carbon source (g/l) 37.5-42.5; MgS047H20 3.75-4.25; CaCI2 3.75-4.25; KH2P04 0.6- 0.8; yeast extract 1.25-1.75; NH4N03 4.25-4.65 & pH 5.0-6.0 to obtain a new growth medium formulation for enhanced production of carotenoids. 2. A process as claimed in claim 1, wherein ratio of C/N ratio is 10. 3. A process for the production of a new growth medium formulation for enhanced production of carotenoids substantially as herein described with reference to the examples.

Full Text

The present invention relates to new growth medium formulation for enhanced production of carotenoids. This invention particularly relates to a growth medium useful for enhanced production of carotenoid by Rhodotorula gracilis.
There has been much interest in the development of new natural colorants for use in food industry. Microbes have recently been recognised as an important source of natural pigments. Rhodotorula, a red yeast produces carotenoid pigments which have great potential for application in food and feed industry both as a colourant and antioxidant in food formulations. The major carotenoid pigments produced by Rhodotorula are p-carotene, torulene and torularhodin. In the present patent, we disclose an innovative formulation of growth medium which effects increase in carotenoid production in comparison with yields in traditional media for yeasts. There are only a few reports on the carotenoids of Rhodotorula yeasts.
Reference may be made to Simpson et al., (1964) who were the first to analyse the various pigments produced by Rhodotorula yeasts and also to propose a biosynthetic pathway for carotenoids (Simpson et al. 1964, J.Bact. 88, p-1688- 1694). The media used by these authors contains 10 % yeast autolysate and 5% glucose (pH 6.0).

Reference may be made to Costa et al., (1989) wherein they have studied the production of carotenoids by Rhodotorula strain and reported to produce 630µg/g of dried cells and the media composition are as follows: (g/1) sucrose 20.0; (NH4)2SO4 3.7; KH2PO4 5.5; Na2HPO4 3.74; MgSO4 7H2O 0.5, yeast extract 1.0, pH 6.5. (Costa et al. 1989, Biotech. Lett., 9, 373-375). The production of carotenoids was noticed in the stationary phase and in non-proliferating conditions.
Reference may also be made to Matelli et al., (1993) wherein they have reported the production of carotenoids by a Rhodotorula strain grown in sugarcane juice containing (g/1) (NH4)2SO4 5.3; KH2PO4 5.5; Na2HPO4 3.7; MgSO4 7H2O 0.5, yeast extract 1.0, pH 6.5 (Matelli et al. J.Fer. Bioeng. 1993). They have found that under non-proliferating conditions the pigment production was 2500 µg/g dried cells.
Reference may also be made to Frengova et al., (1994) wherein a mixed culture of Rhodotorula glutinis and Lactobacillus in whey filtrate were able to produce 268 ug/g dry cells. The media compostion used for carotenoid production is whey filtrate medium containing (g/1) lactose 39.0, (NH4)2SO4 8.0; KH2PO4 3.0; MgSO4 7H2O 0.5, yeast extract 3.0, pH 5.3. Apart from these yeasts, the carotenoids are also produced in plants and many algae especially Dunaliella, which can accumulate 10 % of its

cell dry weight as carotenoids.
The drawbacks connected with hitherto known processes are as follows: In many of the processes using Rhodotomla yeasts the yields of carotenoids were low (268-2500 ug/g of yeast on dry wt basis). Although some of the plants and algal sources produce better yield of carotenoids than Rhodotomla, their cultivation pose many problems. They require sunlight and in some cases high salt concentrations for their growth (e.g., Dunaliella). The quality of carotenoids is not consistent as they are subjected to vagaries of nature. The production of carotenoid pigments canot be easily controlled like in many heterotrophic organisms.
Carotenoids are gaining importance as natural food colorants in view of the possible safety and their synthetic counterparts being considered as harmful to health. Carotenoids function as antioxidants and some of them are precursors for vitamin A. The yield of carotenoids by Rhodotomla yeasts from hitherto known processes were low (268-2500 ug/g yeast on dry wt basis).
The main object of the invention is to provide a process for the production of new growth medium formulation for the production of carotenoids in Rhodotomla which obviates the drawbacks as detailed above.

Another object o the present invention is to check the influence of carbon/nitrogen ratio of media on the production of carotenoids.
Accordingly the present invention provides a process for the production of a new growth medium formulation for enhanced production of carotenoids which comprises mixing of conventional assemblies carbon source (g/l) 37.5-42.5; MgSO47H2O 3.75-4.25; CaCI2 3.75-4.25; KH2P04 0.6-0.8; yeast extract 1.25-1.75; NH4N03 4.25-4.65 & pH 5.0-6.0 to obtain a new growth medium formulation for enhanced production of carotenoids.
The C/N raito of the m3edium is 10 to 40 preferably 10. Rhodotorula gracilis was grown on this medium for 72 h and cells were harvested fro carotenoid estimation.
The maximum total carotenoids production was at 72 h and it was in the range of 25740 to 25990 ug/g dry cells when C/N ratio is 10.
In an embodiment of the present invention carotenoid production was found to occur later than lipid and low C/N ratio being more favourable than high C/N ratio for carotenoid production in Rh. Gracilies.
In an another embodiment of the present invention the high carotenid production was seen in the medium having low pH. Hence the acidic pH of the medium was more suitable for the production of carotenoids.
In yet another embodiment of the present invention the carotenoid.

formation was not found to be associated with the cell growth and the maximum production was reached at the stationary phase.
Rhodotorula gracilis CFR-1 (ATCC 90950) a locally isolated strain of red yeast was grown in different media as given in examples. The organism was grown in Erlenmayer flasks of 500 ml capacity having 250 ml of the above media. The flasks were incubated at 28±2°C on a rotary shaker. The biomass of the culture broth was determined by gravimetry. The cells contain approximately 70% moisture.
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
Rhodotorula gracilis was grown on Minimal salts medium-1 (MSM-1: glucose 40.0, MgSO47H2O 4.0, CaCl2 4.0, KH2PO4 0.75, yeast extract 1.5, NH4NO3 0.286 (g/1), pH 5.5) having a C/N ratio of 160. For comparison this yeast was also grown on the standard YEPD medium (yeast extract-peptone-dextrose (g/1)- yeast extract 10, peptone 20, glucose 20, pH 5.5) and Potato dextrose broth (PDB- (g/1) potato infusion from 200 g, glucose 20, pH 5.5). The carotenoids were estimated at different growth periods like 24, 48, 72, 96 and 120 h and the cells were

harvested by centrifugation for 15 min at 5000 rpm. Cellbiomass was washed with distilled water. 2 g cells were taken in a pestle and mortar and ground with 8 g of sand (acid washed) in the acetone. The pigment was extracted with acetone till all the color was extracted from the cells (30 ml). The acetone extracts were centrifuged at 10,000 rpm for 20 min. Acetone extracts were transferred to 20 ml petroleum ether in a separating funnel and washed with 300 ml of distilled water . The petroleum ether phase was taken in a volumetric flask and the absorbance was read at 485 nm. Carotenoid production occurred very late in the stationary phase in YEPD medium and the total carotenoid production at 120 h was 1610 ug/g dry cells.
EXAMPLE - 2
The organism was grown on Potato dextrose broth (PDB- (g/1) potato infusion from 200 g, glucose 20, pH 5.5) and the carotenoids were estimated at different growth periods like 24, 48, 72 h and the cells were harvested by centrifugation for 15 min at 5000 rpm. Cellbiomass was washed with distilled water. 2 g cells were taken in a pestle and mortar and ground with 8 g of sand (acid washed) in the acetone. The pigment was extracted with acetone till all the color was extracted from the cells (30 ml). The acetone extracts were centrifuged at 10,000 rpm for 20 min.

Acetone extracts were transferred to 20 ml petroleum ether (60-80°C( in a separating funnel and washed with 300 ml of distilled water . The petroleum ether phase was taken in a volumetric flask and the absorbance was read at 485 nm. This medium with complex nitrogen source favoured carotenoid production at very early stationary phase (at 48 h). The total carotenoid production was maximum at 72 h and the yield was 2446 µg/g dry cells.
EXAMPLE-3
The organism was grown on Minimal salts medium-1 (MSM-1: glucose 40.0, MgSO47H2O 4.0, CaCl2 4.0, KH2PO4 0.75, yeast extract 1.5, NH4NO3 0.286 (g/1), pH 5.5) having a C/N ratio of 160 and the carotenoids were estimated at different growth periods like 24, 48, 72, 96 h and the cells were harvested by centrifugation for 15 min at 5000 rpm. Cellbiomass was washed with distilled water. 2 g cells were taken in a pestle and mortar and ground with 8 g of sand (acid washed) in the acetone. The pigment was extracted with acetone till all the color was extracted from the cells (30 ml). The acetone extracts were centrifuged at 10,000 rpm for 20 min. Acetone extracts were transferred to 20 ml petroleum ether (60-80°C) in a separating funnel and washed with 300 ml of distilled water . The petroleum ether phase was taken in a volumetric

flask and the absorbance was read at 485 nm. The carotenoid production starts at 72 h and maximum at 96 h (1796 ug/g dry cells.
The potato dextrose medium with complex nitrogen source favoured carotenoid production at very early stationary phase (at 48 h). The total carotenoid production was maximum at 72 h and the yield was 2446 ug/g dry cells. The carotenoid production in Minimal salts medium-1 with C/N ratio 160 starts at 72 h and was maximum at 96 h (1716 ug/g dry cells).
Table 1. Carotenoid production in different media
(Table Removed)
EXAMPLE - 4
Rhodotorula gracilis was grown on Minimal salts salts medium-2 (MSM-2 (g/1) - glucose 40.0, MgSO47H2O 4.0, CaCl2 4.0, KH2PO4 0.75, yeast extract 1.5, NH4NO3 1.144 pH 5.5) having a C/N ratio of 40. For comparison this yeast was also grown on standard YEPD medium (yeast

extract-peptone-dextrose (g/1)- yeast extract 10, peptone 20, glucose 20, pH 5.5) and Potato dextrose broth (PDB- (g/1) potato infusion from 200 g , glucose 20, pH 5.5). The carotenoids were estimated at different growth periods like 24, 48, 72, 96 and 120 h and the cells were harvested by centrifugation for 15 min at 5000 rpm. Cellbiomass was washed with distilled water. 2 g cells were taken in a pestle and mortar and ground with 8 g of sand (acid washed) in the acetone. The pigment was extracted with acetone till all the color was extracted from the cells (30 ml). The acetone extracts were centrifuged at 10,000 rpm for 20 min. Acetone extracts were transferred to 20 ml petroleum ether in a separating funnel and washed with 300 ml of distilled water . The petroleum ether phase was taken in a volumetric flask and the absorbance was read at 485 nm. Carotenoid production occurred very late in the stationary phase in YEPD medium and the total carotenoid production at 120 h was 1610 ug/g dry cells. The potato dextrose medium with complex nitrogen source favoured carotenoid production at very early stationary phase (at 48 h). The total carotenoid production was maximum at 72 h and the yield was 2446 ug/g dry cells. The carotenoid production in Minimal salts medium -2 starts at 48 h and was maximum at 72 h (2653 ug/g dry cells).

Table 2. Carotenoid production in different media
(Table Removed)
EXAMPLE -5
Rhodotorula gracilis was grown on Minimal salts medium -3 (MSM-3 comprises conventional assimibile carbon source (g/1) 40.0; MgSO47H2O 4.0; CaCl2 4.0; KH2PO4 0.75; yeast extract 1.50; NH4NO3 4.576, pH 5.5) having a C/N ratio of 10. For comparison this yeast was also grown on standard YEPD medium (yeast extract-peptone-dextrose (g/1)- yeast extract 10, peptone 20, glucose 20, pH 5.5) ; Potato dextrose broth (PDB- (g/1) potato infusion from 200 g , glucose 20, pH 5.5). The carotenoids were estimated at different growth periods like 24, 48, 72, 96 and 120 h and the cells were harvested by centrifugation for 15 min at 5000 rpm. Cellbiomass was washed with distilled water. 2 g cells were taken in a pestle and mortar and ground with 8 g of sand (acid washed) in the acetone. The pigment was extracted with acetone till all the color was extracted from the cells (30 ml). The acetone extracts were centrifuged at

10,000 rpm for 20 min. Acetone extracts were transferred to 20 ml petroleum ether in a separating funnel and washed with 300 ml of distilled water . The petroleum ether phase was taken in a volumetric flask and the absorbance was read at 485 nm. Carotenoid production occured very late in the stationary phase in YEPD meium and the total carotenoid production at 120 h was 1610 ug/g dry cells. The potato dextrose medium with complex nitrogen source favoured carotenoid production at very early stationary phase (at 48 h). The total carotenoid production was maximum at 72 h and the yield was 2446 ug/g dry cells. The organism produces maximum amount of carotenoids at 72 h in Minimal salts medium -3 and it was 25990 ug/g dry cells.
Table 3. Carotenoid production in different media
(Table Removed)
The main advantages of the present invention are:
1. A new culture medium for enhanced production of carotenoids by
Rhodotorula gracilis.
2. Medium formulation; Minimal salts medium-3 favours the maximum
production of carotenoid in Rhodotorula gracilis which is more than
10 times higher than the value reported in literature for production of
carotenoids by Rhodotorula.
3. The rapid growth of yeasts cuts production time to a matter of days
and the process lends itself to continuous operation.
4. Compared to plants or animal sources, the present production process
from Rhodotorula gracilis is flexible and can be controlled.
The composition/formulation obtained by the process of the present invention is neither a product of chemical reaction nor a mere admixture but is a synergistic formulation having properties different than the aggregate properties of the individual components

We Claim:
1. A process for the production of a new growth medium formulation for enhanced
production of carotenoids which comprises mixing of conventional assemblies
carbon source (g/l) 37.5-42.5; MgS047H20 3.75-4.25; CaCI2 3.75-4.25; KH2P04 0.6-
0.8; yeast extract 1.25-1.75; NH4N03 4.25-4.65 & pH 5.0-6.0 to obtain a new
growth medium formulation for enhanced production of carotenoids.
2. A process as claimed in claim 1, wherein ratio of C/N ratio is 10.
3. A process for the production of a new growth medium formulation for enhanced
production of carotenoids substantially as herein described with reference to the
examples.

Documents:

1285-del-1999-abstract.pdf

1285-del-1999-claims.pdf

1285-del-1999-correspondence-others.pdf

1285-del-1999-correspondence-po.pdf

1285-del-1999-description (complete).pdf

1285-del-1999-form-1.pdf

1285-del-1999-form-19.pdf

1285-del-1999-form-2.pdf

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Patent Number

215665

Indian Patent Application Number

1285/DEL/1999

PG Journal Number

12/2008

Publication Date

21-Mar-2008

Grant Date

29-Feb-2008

Date of Filing

23-Sep-1999

Name of Patentee

COUNCIL OF SCIENTIFIC AND INDUSTRIAL RESEARCH

Applicant Address

RAFI MARG, NEW DELHI-110001, INDIA.

Inventors:

#	Inventor's Name	Inventor's Address
1	DEYAPPA SOMASHEKAR	FOOD MICROBIOLOGY DEPARTMENT, CENTRAL FOOD TECHNOLOGICAL RESEARCH INSTITUTE, MYSORE-570013
2	RICHARD JOSEPH	393, 3RD STAGE, GOKULUM, MYSORE-570002, KARNATAKA STATE, 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