| PATENT NUMBER | This data is not available for free |
| PATENT GRANT DATE | May 28, 1996 |
| PATENT TITLE |
L-ascorbic acid containing biomass of chlorella pyrenoidosa |
| PATENT ABSTRACT | Microalgal biomass that comprises cells of Chlorella pyrenoidosa which contain greater than 2.0% by dry weight of L-ascorbic acid (Vitamin C), microorganisms and processes that form the biomass, and L-ascorbic acid enhanced animal feed compositions that contain the biomass are disclosed |
| PATENT INVENTORS | This data is not available for free |
| PATENT ASSIGNEE | This data is not available for free |
| PATENT FILE DATE | February 10, 1994 |
| PATENT REFERENCES CITED |
Loewus, F. A., L-Aseorbic Acid: Metabolism, Biosynthesis, Function The Biochemistry of Plants, vol. 3, pp. 77-99 (1980). Vaidya et al., Science and Culture (1971) 37:383-384. Subbulakshmi. et al., Nutirtion Reports International, (1976) 14:581-591. Shigeoka et al., J. Nutr. Sci. Vitaminol (1979) 29:29-307. Shigeoka et al., Agric. Biol. Chem. (1979) 43:2053-2058. Ciferri, Microbiological Reviews (1983) 47:551-578. Gruen and Loewus, Analytical Biochemisty (1983) 130:191-198. McNamer et al, Plant Physiol. (1973) 52:561-564. Reustrom et al., Plant Sci. Lett, 28(1982) pp. 299-305. Aaronson, et al. Arch Microbiol, vol. 112, (1977) pp. 57-59. Vojtisek et al, In "Overproduction of Microbial Metabolites", Butterworths, 1986, pp. 183-198 |
| PATENT PARENT CASE TEXT | This data is not available for free |
| PATENT CLAIMS |
What is claimed is: 1. A culture of Chlorella pyrenoidosa ATCC 53170. 2. A culture of Chlorella pyrenoidosa ATCC 75668. 3. A composition comprising cells of Chlorella pyrenoidosa having all of the identifying characteristics of cells selected from the group consisting of Chlorella pyrenoidosa ATCC 53170 and Chlorella pyrenoidosa ATCC 75668, wherein said cells of Chlorella pyrenoidosa have an intracellular L-ascorbic acid content of at least about 2% by dry weight of said cells. 4. The composition as claimed in claim 3, wherein said cells of Chlorella pyrenoidosa have an intracellular L-ascorbic acid content of at least about 4.0% by dry weight. 5. The composition as claimed in claim 3, wherein said cells of Chlorella pyrenoidosa have all the identifying characteristics of Chlorella pyrenoidosa ATCC 75668 and wherein said cells of Chlorella pyrenoidosa have an intracellular L-ascorbic acid content of at least about 5.0% by dry weight. -------------------------------------------------------------------------------- |
| PATENT DESCRIPTION |
FIELD OF THE INVENTION This invention relates to microalgal biomass that contains a high intracellular level of L-ascorbic acid (Vitamin C), to microorganisms and to processes that form the biomass, and to L-ascorbic acid enhanced animal feed compositions that comprise the biomass. BACKGROUND OF THE INVENTION L-Ascorbic acid, or Vitamin C, is a water-soluble vitamin widely distributed in the plant and animal kingdom. It can be extracted from plant sources, such as paprika, Gladiolus leaves, rose hips, persimmon, and citrus fruit, or synthesized from L-xylose, L-galactose, or D-glucose. F. A. Loewus, L-Ascorbic Acid: Metabolism, Biosynthesis, Function, in The Biochemistry of Plants, Vol. 3, Academic Press, New York, 1980, pp. 77-99, reviews the biosynthesis and sources of L-ascorbic acid. Although most species of animals synthesize L-ascorbic acid, humans and other primates, guinea pigs, fruit eating bats, some birds, and fish such as Coho salmon, rainbow trout channel catfish, and carp cannot. These animals require a dietary source of L-ascorbic acid to prevent scurvy. L-ascorbic acid deficiency in fish also causes scoliosis, lordosis, reduced weight gain, increased susceptibility to bacterial infection, dark skin color, fin erosion, and reduced formation of bone cartilage. In the wild, fish obtain an adequate amount of L-ascorbic acid from aquatic organisms, such as algae. However, fish grown commercially in high density pens or ponds require supplementary L-ascorbic acid to prevent the problems caused by Vitamin C deficiency. L-Ascorbic acid is formulated into fish food before it is pelletized or extruded, but much of it is lost during production of the feed due to the high levels of moisture, heat (135.degree.-175.degree. C.), and pressure used in extrusion process. Even when ethylcellulose-coated L-ascorbic acid is used, about 50% of the L-ascorbic acid is lost. See R. T. Lovell, Trans. Am. Fish Soc., 107, 321-325 (1978). Various algae, including Chlorella species, produce L-ascorbic acid. Aaronson, Arch. Microbiol. 112, 57-59 (1977) discloses light-grown Chlorella that contain up to 15 .mu.g L-ascorbic acid/mg of dry weight of cells, i.e., 1.5% by weight. Renstrom, Plant Sci. Letters, 28, 299-305 (1982/1983) discloses that L-ascorbic acid content in Chlorella is reported to be 6-82 .mu.mol/g dry weight for cells grown in light, i.e., 0.11 to 1.44% by weight. Chlorella pyrenoidosa Chick, culture (UTEX) No. 343, dark-grown for 0.5-6 days in a glucose-enriched medium produced about 3 .mu.mol of L-ascorbic acid per g dry weight (0.06 wt %) and four-fold this amount (0.24 wt %) in 12 hours when light was supplied. However, a need exists for biomass containing higher levels of L-ascorbic acid for use as a dietary supplement. SUMMARY OF THE INVENTION The invention is a microalgal biomass that comprises cells of Chlorella pyrenoidosa, the cells comprising greater than 2.0% by dry weight, preferably greater than 2.5% by dry weight, more preferably greater 4.0% by dry weight, and most preferably greater than 5.0% by dry weight, of L-ascorbic acid. Preferred strains of Chlorella pyrenoidosa are Chlorella pyrenoidosa ATCC 53170 (UV 101-158) and Chlorella pyrenoidosa ATCC 75668 (UV 232-1). DETAILED DESCRIPTION OF THE INVENTION Microorganisms Organisms that produce high levels of L-ascorbic acid have been produced by conventional physical and chemical mutagenizing techniques. Conventional techniques include exposure to radiation, such as ultraviolet irradiation or X-rays, and exposure to a chemical mutating agent, such as, N-methyl-N'-nitro-N-nitrosoquanidine, dimethyl sulfate, ICR 191 (an acridine-based frameshift mutagen), ethyl methane sulfonate, etc. These methods are well know to those skilled in the art. Strains that produce high levels of L-ascorbic acid can be determined with redox dyes. Using analogs of metabolic intermediates to ascorbic acid or inhibitors of the ascorbic acid synthesis, microorganisms may be selected that are capable of maintaining or increasing L-ascorbic acid production in the presence of chemical interference. These progeny may be separated into individual clones and subjected to the procedures repeated to provide microorganisms that produce even higher levels of L-ascorbic acid. Preferred microorganisms are green microalgae of the genus Chlorella, in particular the species Chlorella pyrenoidosa. High L-ascorbic acid-producing strains of Chlorella pyrenoidosa have been derived from such wild strains as Chlorella pyrenoidosa UTEX 1663 and Chlorella pyrenoidosa UTEX 1230. UTEX is the Culture Collection of Algae, Department of Botany, University of Texas at Austin, Austin, Tex., 78713-7640, USA. Cultures are available to the public for a nominal charge, currently $25.00 each. Preferred strains of Chlorella pyrenoidosa are Chlorella pyrenoidosa UV 101-158, which was deposited with the American Type Culture Collection, 12301 Parklawn Drive Rockville, Md. 20852, USA, on Jun. 27, 1985 and given Accession Number 53 170, and Chlorella pyrenoidosa UV232-1, which was deposited with the American Type Culture Collection on Feb. 9, 1994, and given Accession Number 75668. Chlorella pyrenoidosa UV 101-158 was ultimately derived from UTEX 1663 by mutation with ultraviolet light. Chlorella pyrenoidosa UV232-1 was derived in turn from NA28-4, NA19-3, UV165-239, UV137-253. UV101-158, UV29-132, UV18-374, UV12-15, UV3-482, and Chlorella pyrenoidosa UTEX 1663. The prefixes of the mutant strain names indicate the treatment used to create them: C= camphor; DAP=2,6-diaminopurine; EMS=ethyl methanesulfonate; NA=nitrous oxide; DAPN=DAP and NA, in succession; SUV=short wavelength ultraviolet light; UV=broad wavelength ultraviolet light.) Other strains that produce L-ascorbic acid and their parents are as follows. In each case, the mutagenized strain produces more L-ascorbic acid than its parent. DAPN1010-50 was derived from DAP994-1, which was derived, in turn, from C360-70, C284-130, C284-130, C166-4, SUV551-3, DAP389-23, DAP300-5, UV489-5, SUV359-2, SUV296-5, SUV97-1, UV213-4, UV212-11, SUV9-1, UV182-2576, NA5-3, UV137-253, UV101-158, UV29-132, UV18-374, UV12-15, UV3-482, and Chlorella pyrenoidosa UTEX 1663. C166-4 was derived from SUV551-3, which was derived, in turn, from DAP389-23, DAP300-5, UV489-5, SUV359-2, SUV296-5, SUV97-1, UV213-4, UV212-11, SUV9-1, UV182-2576, NA5-3, UV137-253, UV101-158, UV29-132, UV18-374, UV12-15, UV3-482, and Chlorella pyrenoidosa UTEX 1663. NA687-1 and C284-130 were derived from C166-4. NA722-22 was derived from C123-4, which was derived, in turn, from UV609-4, NA528-2 DAP300-6, UV489-5, SUV359-2, SUV296-5, SUV97-1, UV213-4, UV212-11, SUV9-1, UV182 2576, NA5-3, UV137-253, UV101-158, UV29-132, UV18-374, UV12-15, UV3-482, and Chlorella pyrenoidosa UTEX 1663. Biomass Production The process may be a conventional heterotrophic fermentation in which the microorganism is grown under unrestricted growth conditions in a growth-promoting medium at an effective temperature, pressure and pH. The medium contains a suitable carbon source, such as glucose, and dissolved molecular oxygen in an amount sufficient for the cells to grow to a high cell density. Growth is continued until the cells contain the desired amount of L-ascorbic acid. In a preferred process, cells are heterotrophically grown to a high cell density under unrestricted growth conditions. Then, the carbon source is allowed to become substantially depleted so that cell growth substantially ceases. The carbon source is added in restricted amounts so that L-ascorbic acid production continues with substantially no increase in cell density until the L-ascorbic acid concentration reaches the desired level. A sterile aqueous nutrient culture medium is aseptically inoculated with an actively growing culture of the selected microorganism in amounts sufficient to produce, after a reasonable growth period (during which growth is normally exponential), a relatively high cell density. Initial cell densities are generally about 0.15-0.4 g/L, based on the dry weight of the cells. Small amounts of an antifoaming agent may be added initially or during the process as needed. The culture medium includes the carbon source, various salts and, generally, trace metals. For reasons of economy, the carbon source is preferably glucose or a source of glucose. Any saccharide or polysaccharide that can be converted in situ to glucose, e.g. molasses, corn syrup, etc, may be used. The total amount of glucose source used can vary broadly depending upon the particular organism and the result desired. Normally, with high L-ascorbic acid producing organisms, the total amount of glucose source used would, if not metabolized, provide a concentration of about 40-100 g/L typically about 60-85 g/L. In general, the total amount of glucose source used (measured in g/L) is about twice the cell density (g/L, dry weight). Part of the glucose source is normally added initially and the rest during the course of the fermentation. Typically 15-30% of the total glucose is added initially. During the initial glucose source addition and fermentation period the cells are grown to a relatively high density, i.e., 20-50 g/L, typically 30-40 g/L. This permits generation of a high L-ascorbic acid concentration during the carbon-controlled process. The amount of glucose source in the fermentor should be a non-repressing/non-limiting amount. It should optimally promote, but not inhibit or unduly limit, cell growth. Optimum concentrations of the glucose source may vary from organism to organism, and are readily determined by trial for any particular organism. For Chlorella pyrenoidosa strains, glucose source concentrations of 15-30 g/L promote cell growth but do not inhibit growth. Glucose in the supernatant can be determined by the glucose oxidase enzyme test or by high pressure liquid chromatography. When the carbon source concentration drops, it can be replenished as needed. The total concentration should remain below the growth-repressive level, typically about 30 g/L on a glucose-equivalent basis. Desirably, other additives are present initially along with the glucose source. As known in the art, sources of phosphorus, nitrogen, magnesium, iron and trace metals are required. They may be continually or periodically added to the medium, either separately or in conjunction with the glucose source. A typical nutrient medium is described in U.S. Pat. No. 5,001,059. The oxygen source is preferably air, but may be molecular oxygen undiluted or diluted with any gas that is not toxic to the microorganism and unreactive with the fermentation components. The oxygen source is conveniently sparged into the fermentation mass The mass is preferably agitated to distribute the gas throughout the medium and facilitate solution of oxygen therein. The availability of the oxygen in the medium, up to the saturation concentration (100% dissolved oxygen) is largely a function of the agitation rate, flow rate, medium composition, temperature and pressure. The availability of dissolved oxygen is easily controlled by controlling the agitation rate and the flow rate. Depending upon the amount of dissolved oxygen desired at a particular stage of the process, the agitation rate is usually at about 200-1000 rpm and the aeration rate is generally about 0.1-0.6 L of air/minute. During the unrestricted cell growth stage the dissolved oxygen concentration is generally greater than 20% of the saturation value. Preferably, it is 50% or more. The dissolved oxygen concentration is conveniently monitored with an oxygen probe electrode. The temperature and pressure should be such as to promote cell growth and L-ascorbic acid production without destroying the cells. Normally, the temperature is 20.degree.-40.degree. C., preferably about 35.degree. C. The pressure is generally atmospheric but may be superatmospheric. The greater the pressure the greater the solubility of oxygen in the medium. The pH can vary widely depending on the ability of the organism to grow, produce and retain intracellular L-ascorbic acid, i.e. resist secreting it into the aqueous medium. In general, with Chlorella pyrenoidosa and its various strains, the pH is normally in the range of about 6.5 to 8, more usually about 6.9 to 7.5. Relatively high pHs retard passage of L-ascorbic acid from cell to medium. To raise pH, gaseous ammonia gas can be added as needed. Ammonia also serves as a source of nutrient nitrogen. To lower pH, a physiologically compatible acid such as phosphoric, acetic, lactic, or tartaric can be added as needed. Since the microorganisms produce acidic byproducts, pH will decrease if base is not added. During the high oxygen unrestricted growth phase, in which pH is relatively high, intracellular L-ascorbic acid can increase to 1-4 % of the dry weight of the cells. The intracellular L-ascorbic acid content can be increased still further, to at least 2%, preferably greater than 2.5%, more preferably greater 4.0%, and most preferably greater than 5.0% by dry weight of L-ascorbic acid by maintaining the high pH during the low carbon and the low oxygen restricted growth phases. The biomass may be separated from the medium by conventional separation techniques, such as centrifugation, filtration, etc. Following separation, the biomass may be dried by conventional methods, such as, fluid-bed drying, spray drying, drum drying, etc. Care must be taken to minimize air oxidation or thermal degradation of the L-ascorbic acid content. INDUSTRIAL APPLICABILITY Biomass that contains greater than 2.0% by dry weight of L-ascorbic acid is produced. The biomass :may be used as animal feed, either directly, or in admixture as an animal feed composition. Isolation and purification of the ascorbic acid is unnecessary. The biomass can be used as a vitamin C supplement for animals, especially in aquaculture. Biomass provides L-ascorbic acid in a more stable form than is provided by other supplements, especially in fish foods, and at the same time provides other nutrients, such as protein to the animal. The biomass may be fed directly to the animals or mixed with other ingredients to form an animal feed composition. Animal feed compositions for fish typically contain conventional ingredients such as fish meal, soybean meal, distiller's solubles, rice bran and/or hulls, alfalfa meal, peanut meal and/or oil, feather meal, blood meal, and vitamin and mineral supplements. Enriched animal feed compositions typically contain about 60-2,000 g of L-ascorbic acid per ton (907 kg), typically about 320 g/ton |
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| PATENT PHOTOCOPY | Available on request |
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