Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing oxygen-containing organic compound
Reexamination Certificate
2000-01-26
2002-06-11
Lilling, Herbert J. (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing oxygen-containing organic compound
C435S135000, C435S258100, C435S813000, C435S947000
Reexamination Certificate
active
06403345
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention relates to a process for the preparation of highly unsaturated fatty acids, preferably of the physiologically important &ggr;-linolenic acid (all-cis 6,9,12-octadecatrienoic acid; 18:3 n-6; below: “GLA”) from protozoa of the genus Colpidium and to their use.
2. Description of the Prior Art
The interest of the industry in the obtainment and isolation of fatty acids, in particular of fatty acids which are important (essential) in terms of nutritional physiology, preferably polyunsaturated fatty acids, is great. In particular, the development and selection of novel biological sources which could yield GLA inexpensively are worth particular attention. GLA is mainly obtained, depending on resources, from vegetable oils.
In particular, highly unsaturated fatty acids (so-called “PUFA” for: poly-unsaturated fatty acids) are of economical importance, as they have positive effects (1.) as foodstuff additives (in baby foods and many others; see WO 91/11918), (2.) as pharmaceutical active compounds in a large number of indications and (3.) as constituents of cosmetics.
In human and animal metabolism, delta-6 desaturase is the key enzyme for the synthesis of &ggr;-linolenic acid from linoleic acid (18:2 n-6). All other &ohgr;-6 PUFAs and some eicosanoid hormones are derived from GLA. If there is an excessively low activity of the delta-6 desaturase—caused, for example, by age, malnutrition or alcoholism—an undersupply of GLA and its secondary products occurs, as a result of which an number of disorders can be caused.
GLA is therefore used for the human and veterinary treatment of inflammatory and immune diseases (Wu, D., Meydani, S. N., 1996; &ggr;-Linolenic Acid, Metabolism and its role in nutrition and medicine (ed. Huang and Mills), 1996 AOCS, 106-117), in cardiovascular disorders (Deferne, J. L. & Leeds, 1992; J. Hum. Hypertension, 6, 113-119), in particular high blood pressure, diabetes (Horrobin, D. F., 1988; Prog. Lipid Res. 31, 163-194) and certain forms of cancer (Das, U. N, 1996; &ggr;-Linolenic Acid, Metabolism and its role in nutrition and medicine (ed. Huang and Mills), 1996 AOCS, 106-117). The use of GLA is likewise known for the prophylaxis and treatment of chronic, degenerative diseases, in particular rheumatoid arthritis.
Mammals transform GLA into dihomo-GLA (20:3 n-6; DGLA) and concentrate DGLA in mother's milk. In human mother's milk, the content of GLA varies from 0.35 to 1.0% (Gibson, R. A., Kneebone, 1981; Am. J. Clin. Nutr., 34, 252-257). GLA is therefore used in the foodstuffs industry, in particular in infant nutrition.
The provision of native GLA resources takes place especially in higher plants and in a number of microorganisms (Phillips, J. C., Huang, Y.-S., 1996; &ggr;-Linolenic Acid, Metabolism and its role in nutrition and medicine (ed. Huang and Mills), 1996 AOCS, 106-117), such as:
family: Onagraceae: The seeds of the evening primrose (
Oenothera biennis
) contain up to 24% of oil (Whipkey, A., Simon, J. E., Janick, J., 1988; J. Am. Oil Chem. Soc. 65, 979-984); this contains 7-14% of GLA (Wolf, R. B., Kleiman, R., England, R. E., 1983, J. Am. Oil Chem. Soc. 60, 1858-1860). Evening primrose oil is the most frequently used GLA source for clinical and pharmaceutical applications (Horrobin, D. F. 1992; Prog. Lipid Res. 31, 163-194).
family: Boraginaceae: The seed oils of
Borago officinalis
(borage) and
Symphytum officinale
contain a proportion of GLA of 20-27% (Kleiman, R., Earle, F. R., Wolff, I. A., Jones, Q. 1964; J.Am. Oil Chem. Soc. 41, 209-11). Borage oil, however, has higher amounts of longer-chain, monounsaturated fatty acids and contains toxic unsaturated pyrrolidizine alkaloids.
family: Saxifragaceae: The seeds of the blackcurrant (
Ribes nigrum
) contain up to 19% of GLA (Traitler, H., Wille, H. J., Studer, A., 1988; J. Am. Oil Chem. Soc. 65, 755-760).
GLA-containing oils from fermentation of microorganisms are likewise known, such as:
fungi of the genera
Mortierella (
M. ramanniana
, GLA content of the extractable oil about 25%) (Hansson, L., Dostalek, M., 1988; Appl. Microbiol. Biotechnol.: 28, 240-6)
Mucor (
M. rouxii
, and
M. alpina
, GLA content of the extractable oil about 17%) (Lindberg, A. M., Hansson, L., 1991; Appl. Microbiol. Biotechnol., 36, 26-8; Shimitzu, S., Shinmen, Y., Kawashima, H., Akimoto, K., Yamada, H., 1988; Proceedings of ISF-JOCS World Congress 1988, 1000-6);
Phycomycetes (
P. blakesleeanus
, GLA content of the extractable oil about 16%) (Shaw, R., 1965; Biochim. Biophys. Acta, 98, 230-7);
Rhizopus arrhizus
(Kristofikova, L., Rosenberg, M., Vinova, A., Sajbidor, J., Certik, M., 1991; Folia Microbiol., 36, 451-5)
“algae” of the genus
Spirulina (
S. platensis
, GLA content of the extractable oil 12-26%) (Mahajan G., Kamat, M., 1995; Appl. Microbiol. Biotechnol., 43, 466-9; Nichols, B. W., Wood, B. J. B., 1968; Lipids, 3, 46-50),
and also protazoa of the genus
Tetrahymena rostrata
: GLA content of the extractable oil about 21% (according to: Gosselin, Y., Lognay, G., Thonart, P., 1989; Biotechnol. Lett., 11 (6), 423-6)
Tetrahymena thermophila
, GLA content of the extractable oil about 33% (according to Kiy, T., 1993; Dissertation).
Compared with the other biological organisms mentioned, the protozoa are not described much as sources for the obtainment of highly unsaturated fatty acids and to a very great extent are undeveloped for industrial obtainment of GLA.
Their advantages compared with the other known biological sources consist
(1.) in a multiplicity in each case of characteristic fatty acid spectra with, in some cases, a predominant main component in the oil;
(2.) possible culturability in a bioreactor, fermentation;
(3.) culturing which can be controlled accurately during fermentation, i.e. is independent of environmental influences;
(4.) and a defined working-up process following the fermentation;
(5.) if the generation times are significantly shorter than in plants and fungi, such that higher space-time yields result in production.
Fermentation of bacteria, cyanobacteria, algae, fungi and cell cultures from multicellular tissues of plant or animal origin are described in the prior art, but their concentration, working up and purification, e.g. within a fermentation, are not transferable to protozoa.
Fermentation conditions for protozoa (Kiy, Protist, 149, 1998) have only been developed recently. Thus the fermentation of Tetrahymena species is described, which, however, is not transferable to other related species within the protozoa. A disadvantage is moreover the wide fatty acid spectrum with a number of unsaturated fatty acids which are technically complicated to separate (cf.
FIG. 1
) and does not specify the obtainment of GLA. In other protozoa, such as
Paramecium caudatum
and
Colpoda steini
(Dembitskii, V. M., Zharikova, N. I. Inst. Zool. Tolyatti, USSR. Khim. Prir. Soedin. (1998) 2, 294-5), it was only possible to detect traces of GLA.
In the case of the desired industrial production, the impurities of the chosen biological source therefore adversely affect the quality of the GLA-containing biomass obtained. This can bring about an adverse effect on the prophylactic and medicinal action mentioned and therefore necessitates a complex enrichment and purification. Moreover, if the GLA to be obtained is to be employed as a foodstuff and medicament, a biological source is needed for this which is not pathogenic, in particular not a human pathogen.
In addition, it is necessary to establish an economical, inexpensive production together with purification which makes possible industrial utilization.
The object of the present invention is therefore a process for the preparation of GLA from protozoa, in the purest possible enriched form.
SUMMARY OF THE INVENTION
The object is surprisingly achieved by the specific selection of the protozoa of the genus Colpidium, the species
Colpidium campylum
being particularly preferred. Colpidium has a high GLA content in the fatty acid spectrum (cf. FIG.
3
). The GLA fraction is free of contami
Brinkmann Kay
Kiy Thomas
Aventis Research & Technologies GmbH & Co KG
Connolly Bove & Lodge & Hutz LLP
Lilling Herbert J.
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