Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process...
Reexamination Certificate
2001-01-02
2002-11-26
Weber, Jon P. (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Reexamination Certificate
active
06485931
ABSTRACT:
TECHNICAL FIELD
The present invention relates to a method for producing a cholesterol-reduced substance, a cholesterol-reducing composition and a novel cholesterol dehydrogenase, 4-cholesten-3-one dehydrogenase and coprostane-3-one dehydrogenase for using the above-mentioned purpose.
BACKGROUND ART
It is widely known that excess intake of food having high cholesterol content increases the amount of cholesterol in serum and that high cholesterol content in serum is a significant factor in heart diseases. Therefore, processing techniques are required for selectively reducing the amount of cholesterol in food without deteriorating the quality of the food.
Among techniques for reducing the amount of cholesterol in food, a method is known that decomposes cholesterol with microorganisms (Japanese Laid-Open Patent Publication No. 267231/88) as a biochemical technique; however, this method produces by-products, therefore, it is not a safe method. Further, a method in which cholesterol is converted to epicholesterol by using an enzyme is known (WO93/25702).
Beitz, et al., U.S. Pat. No. 4,921,710 describes a method for converting cholesterol to coprostanol by using a cholesterol reductase derived from plants, and suggests a method for converting cholesterol to coprostanol by using a cholesterol reductase derived from bacteria such as
Eubacterium
species ATCC 21408. Also, Beitz, et al., U.S. Pat. No. 5,436,004 describes that the conversion ratio from cholesterol to coprostanol as 0.01% when a cream is treated using the above-mentioned enzyme derived from plants (see, column 5, table 1). However, such a low conversion ratio to coprostanol can not be admitted as practical level.
EYSSEN, British patent 1237483 describes that
Eubacterium
species bacterium separated from feces of rats reduces cholesterol to coprostanol, and also, in EYSSEN, Biochemica et Biophysica Acta, 348,279-284 (1974), it is estimated that the bacterium reduces cholesterol to coprostanol via 4-cholesten-3-one.
Beitz, et al., Applied Microbiology and Biotechnology 43, 887 (1995) describes that
Eubacterium
species bacterium (ATCC 51222) converts cholesterol in micelle to coprostanol, that 4-cholesten-3-one and trace amounts of coprostane have been detected in the conversion process, and that the reduction mechanism of cholesterol using the above-mentioned bacterium might be studied after pure preparations of the cholesterol reductase are obtained.
However, up to now, it has not been confirmed that cholesterol in a substance containing cholesterol is converted via 4-cholesten-3-one and coprostane-3-one to coprostanol by utilizing enzymatic action of cholesterol dehydrogenase, 4-cholesten-3-one dehydrogenase, coprostane-3-on dehydrogenase with using a coenzyme AND (P) and AND (P) H, and enzymes which convert cholesterol in a substance containing cholesterol to coprostanol via 4-cholesten-3-one and coprostane-3-one, respectively, have not been isolated from bacterium which reduces cholesterol, and in addition, no report have been proposed in which food is treated with these converting enzymes and microorganism containing the same.
It is known that cholesterol dehydrogenase derived from
Nocardia, Alcaligenes, Proteus,
which has optimum pH of around 9.0, requires AND (P) as a coenzyme, and is used for a quantitative determination of cholesterol (Japanese Post-Examined Patent Publication No.18064/90), however, this enzyme exhibits low activity at neutral pH, so that it can not be admitted as practical for food treatment.
Testing has been tried to obtain 4-cholesten-3-one dehydrogenase from the feces of a rat, however, the dehydrogenase is rapidly deactivated and can not be purified in this method, therefore, the method can not be admitted as practical (European J. of Biochemistry 37, 143 (1973)).
It is known that treatment of food with a phospholipase, protease and lipase accelerates conversion by a cholesterol oxidase (Japanese Laid-Open Patent publication No.76311/93), however, effect in enzymatic conversion of cholesterol to coprostanol is not known yet.
It is known that meat contains AND (H) (Journal of Food Science 37, 612 (1972)), and that nicotinamide inhibits decomposition of AND (Archives of Biochemistry and Biophisics 156, 143 (1973)). However, addition of nicotinamide in enzymatically converting cholesterol to coprostanol is not known.
It is known that an introduction of the cholesterol oxidase gene into lactic acid bacteria for decomposing food cholesterol (Applied Microbiology and Biotechnology 37, 330 (1992)).
As an applied example of these enzymes, the above-mentioned Beitz, et al., U.S. Pat. No. 5,436,004 suggests a treating method for reducing the amount of cholesterol in serum in which a cholesterol reductase derived from plants is orally administered. Further, it is reported that if a bacterium (ATCC 51222) which reduces cholesterol is orally administered to a rabbit suffering from hypercholesterolemia, the cholesterol level in serum decreases (Letters in Applied Microbiology 20, 137 (1995)).
Since absorbability of coprostanol through the intestinal tract is very low (American J. of Physiology 251, G495 (1986)), conversion of cholesterol to coprostanol is effective as a cholesterol reducing method. A practical method is not known for producing a cholesterol-reduced substance which reduces cholesterol by enzymatically converting cholesterol in food via 4-cholesten-3-one and coprostane-3-one to coprostanol. Also, a method for producing a practical enzyme which can be used in the above-mentioned production method is not known.
DISCLOSURE OF THE INVENTION
The object of the present invention is to provide a novel method for producing a practical cholesterol-reduced substance in food and feed. Another object of the present invention is to provide a novel cholesterol dehydrogenase having optimum pH in neutral pH range which is suitable for the above-mentioned practical method for producing a cholesterol-reduced substance, a novel 4-cholesten-3-one dehydrogenase and coprostane-3-one dehydrogenase having an optimum pH in a weak acidic pH range possessed by meat, and microbial cells and a treated material thereof containing these enzymes. A further object of the present invention is to provide a cholesterol-reducing composition which reduces cholesterol level in serum, comprising the above-mentioned three kinds of novel enzymes or comprising microbial cells containing these novel enzymes.
The present inventors have screened various microorganisms such as stock microorganisms mainly including 300 kinds (species categories) of actinomycetes, fungi and bacteria, aerobic bacteria separated from 100 kinds of various soils, and anaerobic bacteria from 7 feces samples of humans, 8 feces samples of mammals other than human and 9 feces samples of birds, to find a cholesterol reduction converting enzyme group having optimum pH in a neutral pH range or weak acidic range for solving the above-mentioned problems, and have intensively examined a culture medium for enzymatic activity detection and a method for recovering microbial cells, as a result, have accomplished the present invention.
Namely, the present invention relates to a process for producing a cholesterol-reduced substance and a method for reducing the amount of cholesterol, wherein cholesterol in a substance containing cholesterol such as meat, egg, milk, seafood and cooked processed food containing the same, or feed for animals, poultry and pisciculture, and the like, is treated with three kinds of enzymes consisting of cholesterol dehydrogenase having optimum pH in neutral pH range and 4-cholesten-3-one dehydrogenase and coprostan-3-one dehydrogenase having optimum pH in a weak acidic range or microbial cells containing these enzymes, to convert cholesterol to coprostanol for reducing the amount of cholesterol.
Further, the present invention relates to the above-mentioned three kinds of enzymes consisting of cholesterol dehydrogenase having optimum pH in a neutral pH range and 4-cholesten-3-one dehydrogenase and coprostan-3-one dehydrogenase having optimum pH
Aisaka Kazuo
Ando Katsuhiko
Katsumata Ryoichi
Kumazawa Hideyo
Mizukami Toru
Antonelli Terry Stout & Kraus LLP
Kyowa Hakko Kogyo Co. Ltd.
Weber Jon P.
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