Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing oxygen-containing organic compound
Reexamination Certificate
1999-06-25
2004-06-08
Marx, Irene (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing oxygen-containing organic compound
C435S135000, C435S253600, C435S254100
Reexamination Certificate
active
06746857
ABSTRACT:
TECHNICAL FIELD
The present invention relates to a novel culture medium for culturing a microorganism, and a process of producing an unsaturated fatty acid-containing lipid that can be obtained by culturing a microorganism belonging to the genus Mortierella capable of producing unsaturated fatty acids in said medium.
BACKGROUND ART
Arachidonic acid, dihomo-&ggr;-linolenic acid, eicosapentaenoic acid, Mead acid and the like are said to be precursors of prostaglandins, thromboxanes, prostacyclins, leukotrienes and the like that have potent and a variety of biological activities, and thereby are attracting much attention in recent years. For example, a rapid progress has been made on the study of arachidonic acid, as of docosahexaenoic acid (DHA) as a ingredient essential especially for the growth of infants; Lanting et al. have carried out a follow-up study on infants bred with breast milk and those bred with infant formula for three weeks or longer after birth until they grew up to the age of nine years old for the incidence of minor disorders in the cerebral nerves based on their behavioral aspects etc. and have reported that the incidence of cerebral disorders in the children bred with infant formula is twice as high as that of the children bred with breast milk (LANCET, vol. 344, 1319-1322 (1994)).
It has been speculated that this shocking result is due to the possibility that such unsaturated fatty acid as DHA and arachidonic acid that are present in the breast milk but not in the infant formula may be associated with the development of the brain. Since then, many reports have appeared that suggest the association of unsaturated fatty acids with the development of the infant's brain and retina, which is attracting attention as the latest topic in the field of nutrition for preterms and newborns.
These unsaturated fatty acids widely occur in the animal kingdom: for example, arachidonic acid has been isolated from a lipid that was extracted from the adrenal gland or the liver of animals. The content of unsaturated fatty acids therein, however, is low and was insufficient for its large scale supply, and therefore various methods have been devised to obtain unsaturated fatty acids by culturing various microorganisms. Among others, microorganisms belonging to the genus Mortierella are known to produce unsaturated fatty acids such as arachidonic acid, dihomo-&ggr;-linolenic acid, eicosapentaenoic acid, Mead acid and the like and thus methods have been developed that produce said unsaturated fatty acids by the fermentation method using these microorganisms (Japanese Unexamined Patent Publication (Kokai) No. 63(1988)-44891, Japanese Unexamined Patent Publication (Kokai) No. 63(1988)-12290, Japanese Unexamined Patent Publication (Kokai) No. 63(1988)-14696, and Japanese Unexamined Patent Publication (Kokai) No. 63(1988)-14697).
There is also known a method of producing Mead acid using a mutant strain in which the &Dgr;12 desaturating activity has been reduced or defected that can be obtained by subjecting an organism of the genus Mortierella to a mutation treatment (Japanese Unexamined Patent Publication (Kokai) No. 5(1993)-91888). Furthermore, there is also known a method of producing dihomo-&ggr;-linolenic acid using a mutant strain in which the &Dgr;5 desaturating activity has been reduced or defected that can be obtained by subjecting an organism of the genus Mortierella to a mutation treatment (Japanese Unexamined Patent Publication (Kokai) No. 5(1993)-91887).
However, when a fermentation production is carried out in a liquid medium using a filamentous fungus like the genus Mortierella, cellular growth often results in the enhanced viscosity of the liquid culture medium and the ensuing reduced supply of oxygen. Although a method (Japanese Unexamined Patent Publication (Kokai) No. 6(1994)-153970) of regulating dissolved oxygen developed to overcome the above drawbacks has played an important role in enhancing productivity, it is not sufficient to attain high productivity that is economically excellent on an industrial scale. Thereby, the extensive development of culture techniques including the search for more inexpensive culture medium and trace nutrients, the method of regulating mycelial morphology to improve fluidity of the liquid culture medium is imperative.
As a strategy for such technological development, the effect of adding salts as trace nutrients on mycelial morphology are being investigated. There are various reports on effect of adding ions such as potassium, sodium, calcium, magnesium, and phosphoric acid among others (International Application WO96/21037, Japanese Unexamined Patent Publication (Kokai) 8(1996)-214893, Appl. Microbiol. Biotechnol., Vol. 39, p. 450 (1993), Biotechnology Lett., Vol. 12, No. 6, p. 455 (1990), Yukagaku (Oil Chemistry) Vol. 37, No. 3, p. 241 (1989), Yukagaku (Oil Chemistry) Vol. 42, No. 11, p. 893 (1993)). On the other hand, however, there are no reports that investigated the effect of more aggressively enhancing the productivity of unsaturated fatty acids by adding these major ions at concentrations of 0.5 mM or higher exceeding the concept of being nutritional supplements and no reports that even investigated the effects which the balance of added ions has on mycelial morphology and lipid compositions. It is, therefore, desired to optimize the method of adding ions.
DISCLOSURE OF THE INVENTION
Thus, it is an object of the present invention to provide a process of producing a lipid containing unsaturated fatty acids by a fermentation of microorganism belonging to the genus Mortierella, said process comprising adding salts to the culture medium to improve the productivity of unsaturated fatty acids, more specifically all of microbial growth, the accumulation of unsaturated fatty acids, and the accumulation of total lipids, and thereby attaining an economical and stable supply of the lipid containing unsaturated fatty acids. It is also an object of the present invention to provide a culture medium for culturing a microorganism that has an advantage of producing unsaturated fatty acids at high yields and that is inexpensive.
In order to solve the above problems, the present inventors have carried out a comprehensive study concerning the effects of adding salts to a culture medium with respect to not only the yield of unsaturated fatty acids but also changes in mycelial morphology and lipid composition. As a result, the inventors have found that it is very effective to add all the ions of potassium, sodium, calcium, magnesium, and phosphate at defined concentrations in a well-balanced manner and thereby have completed the present invention.
Thus, the present invention provides a culture medium for culturing a microorganism in which phosphate ions, potassium ions, sodium ions, magnesium ions, and calcium ions are in the range of 5 to 60 mM, 5 to 60 mM, 2 to 50 mM, 0.5 to 9 mM, and 0.5 to 12 mM, respectively, and a process having an enhanced productivity of producing unsaturated fatty acids and a lipid containing the same by culturing in said medium a filamentous fungus in particular a microorganism belonging to the genus Mortierella.
The term “unsaturated fatty acids” as used herein refers to the fatty acids having 16 or more carbon atoms and one or more double bonds. Among these, those having 18 or more carbon atoms and two or more double bonds are generally called highly unsaturated fatty acids, which for example include &ggr;-linolenic acid, dihomo-&ggr;-linolenic acid, arachidonic acid, eicosapentaenoic acid, Mead acid, 6,9-octadecadienoic acid, 8,11-eicosadienoic acid and the like.
Embodiment for Carrying Out the Invention
The culture medium for culturing a microorganism of the present invention contains phosphate ions, potassium ions, sodium ions, magnesium ions, and calcium ions in the range of 5 to 60 mM, 5 to 60 mM, 2 to 50 mM, 0.5 to 9 mM, and 0.5 to 12 mM, respectively, preferably in the range of 10 to 45 mM, 10 to 45 mM, 5 to 40 mM, 1 to 6 mM, and 1 to 9 mM, respectively, and can be used for culturing a microorganism for ex
Akimoto Kengo
Higashiyama Kenichi
Shimizu Sakayu
Yaguchi Toshiaki
Marx Irene
Suntory Limited
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