Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing oxygen-containing organic compound
Reexamination Certificate
2000-03-30
2002-06-25
Marx, Irene (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing oxygen-containing organic compound
C435S244000
Reexamination Certificate
active
06410282
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a method for enhancing levels of polyunsaturated fatty acids in thraustochytrid fungi. The present invention particularly relates to a process for enhancement of the polyunsaturated fatty acids, docosahexaenoic acid and eicosapentaenoic acid in cells of microorganisms belonging to the group of fungi termed thraustochytrids, by growing the cells in a medium with increased viscosity. The cells thus enriched in the said polyunsaturated fatty acids (PUFAs) can then be utilized more successfully than cells that are not enriched in the PUFAs, in various beneficial applications that require polyunsaturated fatty acids, such as in animal feeds, human nutrition and extraction of the PUFAs for nutritional supplementation.
BACKGROUND OF THE INVENTION
Fatty acids are constituents of lipids, which are required by all living organisms for growth, survival and reproduction. Among the fatty acids, saturated fatty acids are those with a chemical structure in which the carbon atoms are connected to each other only by single bonds and contain no double bonds. Unsaturated fatty acids are those in which one or more of the carbon atoms are connected to each other by double bonds. Polyunsaturated fatty acids, termed as PUFAs hereafter, are those in which more than one such double bonds are found.
Among the PUFAs, two are considered extremely essential in the health of animals and human beings. These are the docosahexaenoic acid and eicosapentaenoic acid, termed DHA and EPA hereafter. The molecular structure of both DHA and EPA is such that the first double bond follows the third carbon atom from the methyl end of the fatty acid structure. Therefore, these are also called omega-3 PUFAs. DHA contains 22 carbon atoms, between which six double bonds are found. EPA contains 20 carbon atoms, between which five double bonds occur. Both DHA and EPA have been shown to be important for human health and in animal nutrition. In human health, DHA and EPA have been shown to be important in brain development in children, prevention of atherosclerosis, prevention of night blindness, neurological disorders and even for possible prevention of cancer (Bajpai, P. and P. K. Bajpai. 1993. Journal of Biotechnology 30: 161-183; Barclay, W. R. et al. 1994. Journal of Applied Phycology 6: 123-129; U.S. Pat. No. 9,428,913; Singh, A. and O. P. Ward. 1997. Advances in applied microbiology, 45: 271-312). These two omega-3 PUFAs have been shown to enhance growth and reproduction in crustacean animals, such as prawns, which are very important as aquaculture animals for human consumption (Harrison, K. E. 1990. Journal of Shellfish Research 9: 1-28). Incorporation of DHA and EPA in human and animal feeds is therefore considered important. DHA and EPA levels of thraustochytrid fungi can be enhanced beyond their natural levels by growing the cells in a medium with increased viscosity, as detailed in the present invention, and their cells can be of still better use as supplement to human nutrition and as feed for animals compared to presently known processes. Thraustochytrids can be cultivated on a large scale, using well established fermentation techniques. Cells thus obtained can be used as animal feeds, by suitably processing and preserving their cells, such as by spray-drying and freezing. The cell biomass, enhanced in the omega-3 fatty acids can also be harvested and DHA and EPA extracted in a pure form. These may be used to supplement human food that is poor in these essential omega-3 PUFAs.
One major source of EPA and DHA for human consumption is in the form of fish oil. However, fish oil has the disadvantage of an odour, which is disagreeable to many human consumers. Fish containing DHA and EPA are also highly seasonal and variable in their omega-3 PUFA contents. Besides, most of the fish oil is hydrogenated and the omega-3 PUFAs are destroyed. For these reasons, micro-organisms containing EPA and DHA, which can be cultivated on a large scale are considered suitable for use in human nutrition and animal feeds (Bajpai, P. and P. K. Bajpai. 1993. Journal of Biotechnology 30: 161-183). Several single-celled plants, the algae, contain high levels of EPA and DHA and have been considered for the said purposes. References may be made to D. L. Alonso et al. (Alonso, D. L. et al., 1992. Aquaculture 102: 363-371). However, large scale cultivation of these plants in natural ponds often is subject to the problem of other microorganisms growing along with these plants. This may pose a health problem to human consumers. Growing them in pure cultures in fermentors is cost-intensive, since these plants require light and suitable photo reactors are very expensive to maintain and operate. Microorganisms can be easily cultivated on a large scale using cheap nutrients. Several groups of microorganisms contain high amounts of EPA and DHA. Such organisms can be used directly as feed, or the said PUFAs can be extracted from them for further use. Search for microorganisms containing high amounts of DHA and EPA has shown that thraustochytrid fungi contain some of the highest amounts of DHA and EPA. Thraustochytrids are already considered of commercial importance. Their cells are used in animal feeds or for extraction of PUFAs for commercial use (Singh, A. and O. P. Ward. 1997. Advances in applied Microbiology 45: 271-312). The Japanese Patent No. 9633263 (1996) describes a strain of a thraustochytrid for application in the food industry such as food-additives, nutritional supplements, as additives for infant milk formula, feedstuffs and drug additives. The strain contains at least 2% of dry wt as DHA. Another Japanese patent No. 980 3671 (1998) describes the production by fermentation of DHA and another PUFA, docosapentaenoic acid (DPA) from lipids of thraustochytrid fungi. U.S. Pat. No. 5,340,594 describes a process for production of whole-celled or extracted microbial products using thraustochytrid fungi with a high concentration of the omega-3 PUFAs. U.S. Pat. No. 5,340,742 discloses a process for growing the thraustochytrid fungi in defined media suitable for their growth. All the above patents relate to screening numerous thraustochytrid cultures, selecting the strain with the highest DHA and EPA content, prepare mutant strains of these and cultivate such strains under optimal culture conditions for commercial application.
The present invention aims to further increase the DHA and EPA levels in thraustochytrid fungi so that they will provide still higher commercial yields of the said PUFAs. Besides, the above mentioned prior art patents reject a large number of strains, which might have only moderate DHA and EPA concentrations. In the present invention, even strains with moderate amounts of DHA and EPA can be made to produce large amounts of these PUFAs by growing them in a medium with increased viscosity. Strains that naturally have high concentrations of DHA and EPA can be made to produce even more of these using the present process.
OBJECTS OF THE INVENTION
The main object of the present invention is to enhance the amounts of PUFAs in thraustochytrid fungi, which obviates the drawbacks as detailed above.
Another object of the invention is to make strains of thraustochytrids to produce higher amounts of DHA and EPA than they normally produce using optimal nutrient conditions.
Yet another object of the present invention is to enhance the levels of these fatty acids by growing the cultures of thraustochytrid fungi in a medium with increased viscosity.
SUMMARY OF THE INVENTION
To meet the above objects, the present invention provides a method for enhancing levels of polyunsaturated fatty acid levels in thraustochytrid fungi, using culture media supplemented with polyvinyl pyrrolidone (PVP) to increase viscosity.
DETAILED DESCRIPTION OF THE INVENTION
Accordingly, the present invention provides a method for enhancing levels of polyunsaturated fatty acid levels in thraustochytrid fungi, using culture media supplemented with polyvinyl pyrrolidone (PVP) to increase viscosity and which comprises: S
Chandramohan Dorai Rajasingam
Desa Ehrlich
Kumar Seshagiri Raghu
Council of Scientific and Industrial Research
Foley & Lardner
Marx Irene
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