Title of Invention

A NOVEL CULTURE MEDIA FOR THE PRODUCTION OF CAROTENOIDS USING BACTERIAL SPECIES ABCC001 OF GENUS PARACOCCUS

Abstract

Title: A NOVEL CULTURE MEDIA FOR THE PRODUCTION OF CAROTENOIDS USING BACTERIAL SPECIES ABCCOOl OF GENUS Paracoccus The present invention relates to a novel culture media for the production of carotenoids using bacterial species ABCCOOl (having depository accession no. MTCC 5248) of genus Paracoccus comprises carbon source 2 - 20 gm per 1 liter of culture medium; nitrogen source 1-7 gm per 1 liter of culture medium; inorganic salts 0.001 - 1 gm per 1 liter of culture medium; complex nutrients like vitamins, nucleic acids, yeast extract, peptone, meat extract, malt extract 0.2 - 10 gm per 1 liter of culture medium during one life cycle. Bacterial species ABCCOOl produces either mixed carotenoids or single dominant carotenoid and minor carotenoids inside the cells during its growth phase. The bacterial species ABCCOOl is isolated from the local soil.

Full Text

The present invention relates to a novel f p. 7, for the production of carotenoids using bacterial specie genus II Paracoccus. nhshqcf The other objective of the present inventio carotenoid pigments from the bacterial strain ABCCOOl us ilture media for the growth of bacteria. Bacterial strain produces asia a, p-carotene, canthaxanthin, a-carotene etc. The Identification Reference Paracoccus sp. ABCCOOl has been deposited at Microbial Type Culture Collection & Gene Bank on 12/08/2008 and Accession Number given by the International Depositary Authority is MTCC 5248. BACKGROUND OF THE INVENTION: Carotenoids are natural pigments that are present in living organisms in yellow, orange and red colours. Among the living organisms, carotenoids are present in plants and microorganisms like bacteria, algae and fungi. The carotenoids have two main biological functions namely light harvesting pigment in photosynthesis and protect against photo-oxidative damage. In bacteria having carotenoids, these regulate the illumination for photosynthesis. As part of the light-harvesting antenna, carotenoids can absorb photons and transfer the energy to chlorophyll, thus assisting in the harvesting of light in the range of 450 - 570 nm (Cogdell EJ and Frank HA 1987). Apart from Photosynthetic bacteria, a few heterotrophic bacteria also produce yellow, orange and red pigment carotenoids. Flavobacterium sp. is reported as carotenoids producing bacteria in early 70's and later it is identified as a new genera, Paracoccus sp. in the late 90's. Presently eight species are recognized within the genus Paracoccus: P. denitrificans (the type species of the genus) [van Verseveld, H. W., and A. H. Stouthamer. 1992. The genus, Paracoccus, p. 2321-2334. In A. Balows, H. G. Truper, M. Dworkin, and K.H. Schleifer (ed.), prokaryotes. A handbook on the biology of bacteria: ecophysiology, isolation, identification, applications, 2nd. ed., vol. 3. Springer-Verlag, New York; and Visuvanathan, S., M. T. Moss, J. L. Stanford, J. Hermon-Taylor, and J. J. McFadden. 1989. Simple enzymatic method for isolation of DNA from diverse bacteria. J. Microbiol. Meth. 10:59-64], P. thiocyanatus [Katayama, Y., A. Hiraishi, and H. Kuraishi. 1995. Paracoccus thiocyanatus sp. nov., a novel species of thiocyanate-utilizing facultative chemolithotroph, and transfer of Thiobacillus versutus to the genus Paracoccus as Paracoccus versutus comb. nov. with emendation of the genus. Microbiology 141:1469-1477], P. versutus (formerly known as Thiobacillus versulus) [Katayama, Y., A. Hiraishi, and H. Kuraishi. 1995. Paracoccus thiocyanatus sp. nov., anew species of thiocyanate-utilizing facultative chemolithotroph, and transfer of Thiobacillus versutus to the genus Paracoccus as Paracoccus versutus comb. nov. with emendation of the genus. Microbiology 141:1469-1477], P. kocurii [Ohara, M., Y. Katayama, M. Tsuzaki, S. Nakamoto, and H. Kuraishi. 1990. Paracoccus kocurii sp. nov., a tetramethylammonium-assimilating bacterium. Int. J. Syst. Bacteriol. 40:292-296], P. alcaliphilus [Urakami, T., J. Tamaoka, K. Suzuki, and K. Komagata. 1989. Paracoccus alcaliphilus sp. nov., an alkaliphilic and facultatively methylotrophic bacterium. Int. J. Syst. Bacteriol. 39:116-121], P. aminophilus, P. aminovorans [Urakami, T., H. Araki, H. Oyanagi, K. Suzuki, and K. Komagata. 1990. Paracoccus aminophilus sp. nov. and Paracoccus aminovorans sp. nov., which utilize N,N'-dimethylformamide. Int. J. Syst. Bacteriol. 40:287-291], and P. solventivorans [Siller, H., F. A. Rainey, E. Stackebrandt, and J. Winter. 1996. Isolation and characterization of a new gram-negative, acetone degrading, nitrate-reducing bacterium from soil, Paracoccus solventivorans sp. nov. Int. J. Syst. Bacteriol. 46:1125-1130]. The strain previously known as P. halodenitrificans has recently been transferred to the genus Halomonas on account of its phylogenetic affiliation [Dobson, S. J., and P. D. Franzmann. 1996. Unification of the genera Deleya (Baumann et al. 1983), Halomonas (Vreeland et al. 1980), and Halovibrio (Fendrich 1988) and the species Paracoccus denitrificans (Robinson and Gibbons 1952) into a single genus, Halomonas, and placement of the genus Zymnobacter in the family Halomonadaceae. Int. J. Syst. Bacteriol. 46:550-558]. None of the eight known members of the Paracoccus genus produces carotenoids. The US Patent 5,935,808 relates to a novel carotenoid producing bacterial species type strain DSM 11574 which produces and secretes carotenoids in vesicles and a process for production of carotenoids comprising (a) culturing a bacterial species in an nutrient medium including sources of carbon, nitrogen and inorganic substances; and (b) recovering an individual carotenoid pigment or a mixture of carotenoid pigments from the cells, vesicles and/or medium and process for production of carotenoids using the same. (Prior application No.08/902,518, filed in July 29, 1997 now US patent 5,935,808). This strain is identified as Paracoccus marcusii MH I based on morphological, biological, biochemical assays and determined by 16S ribosomal RNA analysis. This patent specified that the said strain DSM 11574 as a gram-negative brightly orange coccoid bacterium that appeared as a contaminant on a nutrient agar plate as isolated. The phenotypic characterization and phylogenetic analysis based on 16S rDNA sequence comparisons showed that the bacterium so isolated should be classified as a hitherto unknown species within the genus, Paracoccus with the proposed name Paracoccus marcusii sp. This invention further provided a process for production of carotenoids using a bacterial species secreting same as vesicles to the growth medium and extraction of the carotenoids from the vesicles. The term "vesicle" as used herein in the specification refers to any substantially globular lipophillic body which is not a life form, i.e., is not capable of reproduction. It is also observed that in respect of the said species and strain, when the NaCl concentration in the process medium is increased to 6 g per liter, growth is slow, and above 8 g per liter of NaCl no growth is obtained and that Nitrate did not support anaerobic growth, and is * not reduced to nitrite. DISCLOSURE OF THE INVENTION: The present invention elucidates a novel culture media for the production of carotenoids using bacterial species ABCCOOl which is most similar to bacteria of the Paracoccus genus based on biochemical characterization. The Identification Reference Paracoccus sp. ABCCOOl has been deposited at Microbial Type Culture Collection & Gene Bank on 12/08/2008 and Accession Number given by the International Depositary Authority is MTCC 5248. The bacterial species produces carotenoids like astaxanthin, P-carotene, canthaxanthin, adinoxanthin, a-carotene etc. Eight distinct species are presently associated with the Paracoccus genus and none other than that stipulated as an agar plate contaminant in US Patent 5935808, produce carotenoids. There is however no known species of this genus isolated from soil that produces carotenoids during its life cycle. The present invention provides a novel media for production of mixed carotenoids and/or single dominant carotenoid and minor carotenoids. The bacterial species ABCCOOl accumulates both primary and secondary carotenoids simultaneously as a mixed carotenoid profile. The bacterial species ABCCOOl accumulates single dominant carotenoids such as astaxanthin, P-carotene , canthaxanthin, adinoxanthin, a-carotene etc. The single dominant carotenoid betacarotene is produced in all its isomers and astaxanthin is accumulated in its free forms only. Out of all the bacterial genera presently known, the bacterial species used in the present invention is most similar to the Paracoccus genus, based on biochemical studies. This species producing carotenoid is a local soil isolate which is similar to the bacteria Paracoccus marcusii MHl isolated as contaminant in nutrient agar plate. The bacterial species ABCCOOl used in the present invention produces carotenoid inside the cell and is not secreted out as vesicles to the growth medium. In another aspect, the present invention differ from the carotenoid producing and secreting bacterial species by the following phenotypic and biochemical characterization as follows: Description P. marcusii MHl ABCCOOl Isolation Nutrient agar plate contaminant Soil isolates Colony on Agar Smooth, flat, brightly orange colored Smooth or dry, Opaque, brightly orange colored Optimum temperature 25-30°C 28-40°C Growth on NaCl at 8g and above per liter No growth Luxuriant Nitrate reduction to nitrite -ve +ve Carotenoid secretion as vesicles Yes No The bacterial species ABCCOOl is however similar to the genus Paracoccus in that it consists of gram-negative cocci or short rods, showing a substantial metabolic versatility. Representatives are able to grow aerobically on a wide range of organic compounds. Medium for production of carotenoids using the bacterial species ABCCOOl contains a carbon source, a nitrogen source and inorganic salts necessary for the growth of producer microorganisms, as well as if necessary special required substances (for example, vitamins, amino acids, nucleic acids etc.). Various carbon sources, sugars such as glucose, sucrose, lactose, fructose, trehalose, mannose, mannitol, maltose, etc., added from 2 to 20 g per 1 liter in the process medium. Various nitrogen sources of organic and inorganic nitrogen are used, like potassium nitrate, ammonium nitrate, ammonium chloride, ammonium sulfate, ammonium phosphate, ammonia, urea, Sodium glutamate etc., are used alone or in combination. The concentration of the nitrogen source added is varied according to the kind of the nitrogen source, preferably 1 to 7 g per 1 liter medium. Various inorganic salts like potassium dihydrogen phosphate, dipotassium hydrogen phosphate, magnesium sulfate, magnesium chloride, ferrous sulfate, ferrous chloride, zinc sulfate, zinc chloride, cupric sulfate, calcium chloride, calcium carbonate, sodium carbonate, etc., may be used alone or in combination. The concentration of inorganic acid varies according to the kind of the inorganic salt from 0.001 to 1 g per 1 liter medium. Various complex nutrient ingredients like vitamins, nucleic acids, yeast extract, peptone, meat extract, malt extract, etc., are used in combination or alone at a concentration from 0.2 g to lOg, per 1 liter medium. The following examples, which together with the above descriptions illustrate the invention. EXAMPLES The following protocols and experiments carried out in this invention are: Example 1: Source of organism: ABCCOOl appeared, as brightly orange colony in nutrient agar plate is isolated from soil. The bacterial strain, ABCCOOl is identified as Paracoccus sp. based on biochemical characterization. Media and culture condition: The ABCCOOl is grown at room temperature varied in the seasons from (28°C - 40°C) in medium containing 5g of bacto-peptone, 3 gram of yeast extract and 5g of NaCl at pH 7.0. Liquid culture is grown under shaking condition at 140 rpm. The composition of medium is modified based on the requirement of nutrient to produce one specific biologically active carotenoid. Microscopy: Cultures are observed in Labo standard microscope equipped with phase contrast. Physiological and biochemical characterization: Test for identification of the ABCCOOl is carried out, using standard methods as described in The Prokaryotes and Bergey's manual of systemic bacteriology. Selected physiological tests are determined with Active Pharmaceutical Ingredients (API) for metabolic reaction, and aerobic growth on different carbon sources. All the API tests are performed under the direction of the manufacturer. Experimental analysis on Carotenoid production: Aliquots of ABCCOOl are harvested by centrifugation at 13000x'g' for 10 min and washed thrice with saline water. The harvested cells are re-suspended with the solvent of acetone/ethyl acetate/ methanol/ethanol and extracted by milling in motor and pestle with acidified sand until the acetone turned colourless. The extract is filtered through 0.4|j, size nitrocellulose filter paper. The filtered extract is concentrated in rotovapour under vacuum. The pigment is blown to dryness under a stream of N2 and stored at -20°C until analysis. Carotenoid profile of the ABCCOOl is analysed in HPLC, Jasco, Japan, using Supleco column (25.0cm x 0.46cm) with a 5cm guard column at A455 and a Mobile Phase of Acetonitrile: Methanol: Ammonium acetate (0.2%): Ethyl Disopropanolamine: n- Propanol: BHT (455:500:25:200 ^il: 20 ml: 50mg) at a flow rate of 1.3 ml per minute. The sample is injected in 20|il aliquots via on-line rheodyne injector unit. A diode-array detector is used to monitor spectra on line and to integrate chromatograms. Chiralitv Configuration: Chirality's configuration of astaxanthin and betacarotene is determined by HPLC along with the traces of canthoxanthin, and a-carotene. The respective carotenoids are determined with standards obtained from Sigma, USA. Morphological features of bacterial species ABCCOOl of genus Paracoccus Bacterial species ABCCOOl formed cocci to short rods, 1 to 2 by 1 to 1.5 mm size. It consisted mainly of pairs and short chains or clusters of up to 4 - 5 bacteria. Cells are non-motile and do not form spores. Bacterial species ABCCOOl stained gram-negative. Colonies on agar are smooth, opaque and brightly orange coloured. Physiological and biochemical characterization of bacterial species ABCCOOl of %QmiLS Paracoccus: Optimum temperature for growth is 25-37° C. Doubling time in the standard growth medium at the optimal temperature is 2.30 hours. Luxuriant growth is found, while the NaCl concentration is increased up to 8 g per liter. The following carbon and energy sources could be used for growth: D-glucose, D-fructose, D-galactose, D-mannose, L-arabinose, maltose, cellobiose, D-lactose, melibiose, sucrose, turanose, D- trehalose, gentobiose, lactulose, glycerol, D-sorbitol, xylitol, m-inositol, adonitol, D-arabitol, propionic acid, cis-aconitic acid, citric acid, DL-lactic acid, malonic acid, quinic acid, succinic acid, malic acid, formic acid, L-alanine, and alaninamide. No growth is obtained on L-flicose, D-psicose, L-rhamnose, raffinose, dextrin, glucose-1-phosphate, glucose-6-phosphate, methonal, glycine, D-alanine, L-alanyglycine, L-asparagine, L-aspartate, L-glutamate, L-histidine, L-lucine, L-omithine, L-phenylalanine, L-proline, L-serine, inosine, uridine and thymidine. Biochemical Interpretation of bacterial strain ABCCOOl: It is obligatory aerobic. Cytochrome oxidase and catalase reactions shows positive. Nitrate does not support anaerobic growth and glucose is not fermented. Nitrate is reduced to nitrite. Starch and Gelatin are not hydrolysed. Arginine di hydrolysed and urease activities are not detected. Indole negative, citrate utilization is negative and MRVP test negative. We claim: 1. A novel culture media for the production of carotenoids using bacterial species ABCCOOl of genus Paracoccus comprises carbon source 2 - 20 gm per 1 liter of culture medium; nitrogen source 1 - 7 gm per 1 liter of culture medium; inorganic salts 0.001 - 1 gm per 1 liter of culture mediimi; complex nutrients like vitamins, nucleic acids, yeast extract, peptone, meat extract, malt extract 0.2 - 10 gm per 1 liter of culture medium during one life cycle. 2. A novel culture media using bacterial species ABCCOOl of genus Paracoccus as claimed in claim 1 producing either mixed carotenoids or single dominant carotenoid and minor carotenoids. 3. A novel culture media as claimed in claim 1 wherein bacterial species ABCCOOl of genus Paracoccus is characterized as a non-vesicular bacterium accumulating colony morphology on nutrient agar; salinity tolerance from 0.2 to 1.0%; exhibits reduction of nitrate to nitrite. We claim: 1. A novel culture media for the production of carotenoids using bacterial species ABCCOOl (having depository Accession No. MTCC 5248) of genus Paracoccus comprises carbon source 2 - 20 gm per 1 liter of culture medium; nitrogen source 1 - 7 gm per 1 liter of culture medium; inorganic salts 0.001 - 1 gm per 1 liter of culture medium; complex nutrients like vitamins, nucleic acids, yeast extract, peptone, meat extract, malt extract 0.2-10 gm per 1 liter of culture medium during one life cycle. 2. A novel culture media using bacterial species ABCCOOl (having depository Accession No. MTCC 5248) of genus Paracoccus as claimed in claim 1 producing either mixed carotenoids or single dominant carotenoid and minor carotenoids. 3. A novel culture media as claimed in claim 1 wherein bacterial species ABCCOOl (having depository Accession No. MTCC 5248) of genus Paracoccus is characterized as a non-vesicular bacterium accumulating colony morphology on nutrient agar; salinity tolerance from 0.2 to 1.0%; exhibits reduction of nitrate to nitrite. 4. A novel culture media as claimed in claim 1 wherein bacterial species ABCCOOl (having depository Accession No. MTCC 5248) of genus Paracoccus is isolated from the soil. 5. A novel culture media as claimed in claim 1 and 2 wherein either mixed carotenoids or single dominant carotenoid produce inside the cells. 6. A novel culture media for the production of carotenoids using bacterial species ABCCOOl (having depository Accession No. MTCC 5248) of genus Paracoccus as claimed in claim 1 and 2 wherein the bacterial species ABCCOOl (having depository Accession No. MTCC 5248) accumulates carotenoids during its growth phase. 7. A novel culture media for the production of carotenoids using bacterial species ABCCOOl (having depository Accession No. MTCC 5248) of genus Paracoccus as claimed in claim 6 wherein the bacterial species ABCCOOl (having depository Accession No. MTCC 5248) accumulates both primary and secondary carotenoids simultaneously as a mixed carotenoid profile. 8. A novel culture media for the production of carotenoids using bacterial species ABCCOOl (having depository Accession No. MTCC 5248) of genus Paracoccus as claimed in claim 6 wherein the bacterial species ABCCOOl (having depository Accession No. MTCC 5248) accumulates predominately a single dominant carotenoid. 9. A novel culture media for the production of carotenoids using bacterial species ABCCOOl (having depository Accession No. MTCC 5248) of genus Paracoccus as claimed in claim 8 wherein the said single dominant carotenoid is either astaxanthin, P-carotene, canthaxanthin, adinoxanthin or a-carotene. 10.A novel culture media for the production of carotenoids using bacterial species ABCCOOl (having depository Accession No. MTCC 5248) of genus Paracoccus as claimed in claim 8 wherein the single dominant carotenoid betacarotene is produced in all its isomers such as 9 cis, di cis and trans isomers. 11.A novel culture media for the production of carotenoids using bacterial species ABCCOOl (having depository Accession No. MTCC 5248) of genus Paracoccus as claimed in claim 8 wherein the single dominant carotenoid astaxanthin is accumulated in its free forms only. 12.A novel culture media for the production of carotenoids using bacterial species ABCCOOl (having depository Accession No. MTCC 5248) of genus Paracoccus as claimed in claim 1 wherein the source of carbon are glucose, sucrose, lactose, fructose, trehalose, mannose, mannitol, maltose. 13.A novel culture media for the production of carotenoids using bacterial species ABCCOOl (having depository Accession No. MTCC 5248) of genus Paracoccus as claimed in claim 1 wherein source of organic and inorganic forms of nitrogen are potassium nitrate, ammonium nitrate, ammonium chloride, ammonium sulfate, ammonium phosphate, ammonia, urea. Sodium glutamate. 14.A novel culture media for the production of carotenoids using bacterial species ABCCOOl (having depository Accession No. MTCC 5248) of genus Paracoccus as claimed in claim 1 wherein source of inorganic salts are potassium dihydrogen phosphate, dipotassium hydrogen phosphate, magnesium sulfate, magnesium chloride, ferrous sulfate, ferrous chloride, zinc sulfate, zinc chloride, cupric sulfate, calcium chloride, calcium carbonate, sodium carbonate. 15.A novel culture media for the production of carotenoids from the bacterial species ABCCOOl (having depository Accession No. MTCC 5248) of genus Paracoccus as claimed in claim 1 wherein concentration of NaCl is 8 gm or above per liter of culture media. 16.A novel culture media for the production of carotenoids from the bacterial strain ABCCOOl (having depository Accession No. MTCC 5248) as claimed in claim 1 wherein the optimum temperature used for the cultivation of bacterial species ABCCOOl (having depository Accession No. MTCC 5248) is between 28° C to 40° C. 17.A novel culture media for the production of carotenoids using bacterial species ABCCOOl (having depository on Accession No. MTCC 5248) of genus Paracoccus is substantially herein described with foregoing description and examples.

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0860-mas-2002 abstract.pdf

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0860-mas-2002 claims.pdf

0860-mas-2002 correspondence-others.pdf

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0860-mas-2002 description (complete) duplicate.pdf

0860-mas-2002 description (complete).pdf

0860-mas-2002 description (provisional).pdf

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0860-mas-2002 form-19.pdf

0860-mas-2002 form-26.pdf

0860-mas-2002 form-3.pdf

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860- mas-2002 complete specification as granted.pdf