Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
2001-07-17
2004-01-13
Marx, Irene (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S252500, C435S133000, C424S442000, C426S061000
Reexamination Certificate
active
06677141
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to a method for culturing
Bacillus subtilis
, the cultured microorganism obtained by the method, a water-soluble vitamin K derivative originating in the cultured microorganism, and a food product, beverage, or feed containing the cultured microorganism or the water-soluble vitamin K derivative. More particularly, this invention relates to a method for culturing
Bacillus subtilis
in such a manner as to induce storage of a vitamin K derivative, particularly a menaquinone-7 (vitamin K
2
) derivative, in the largest amount within the cells of the
Bacillus subtilis
, a cultured product of
Bacillus subtilis
having a vitamin K derivative, particularly a menaquinone-7 (vitamin K
2
) derivative, accumulated in a large amount within the cells thereof in consequence of the culture by the method mentioned above, a water-soluble vitamin K derivative, particularly a water-soluble menaquinone-7 (vitamin K
2
) derivative, originating in the cultured product mentioned above, and a food product, beverage, or feed containing the cultured product or the water-soluble vitamin K derivative, particularly the water-soluble menaquinone-7 (vitamin K
2
) derivative.
2. Description of the Related Art
Vitamin K has been heretofore known as a factor found necessary during the coagulation of blood. Since the deficiency of this vitamin brings deterioration of the ability to coagulate blood, vitamin K is held to constitute itself one species of fat-soluble vitamin otherwise called an antihemorrhagic vitamin. As regards the cause for the deterioration of ability to coagulate blood thus brought about by the deficiency in this vitamin K, it has been suggested in recent years that vitamin K is indispensable for the biosynthesis of several blood coagulation factors including prothrombin. The amount of the vitamin K which is found necessary for the purpose of preventing the deterioration of the blood-coagulating ability is extremely small, i.e. on the order of &mgr;g. Generally, since adults are supplied with this vitamin K by enterobacteria, they rarely contract the disease of vitamin K deficiency. The synthetic vitamins K
1
and K
2
are used as medicines for the therapy of the disease of the vitamin K deficiency hemorrhage. To date, the vitamin K has not attracted much attention because the concentrate of natural vitamin K
1
has been utilized heretofore in the form of a food product for the purpose of preventing this disease.
It has been demonstrated in recent years, however, that vitamin K has a function of promoting ossification and a function of repressing bone resorption and that the density of bone is increased by the administration of the vitamin K. The osteoporosis is the pathosis of embrittlement of bone caused by senility or illness. Since this disease accompanies fracture and severe pain, it has been posing a serious social issue from the standpoint of geriatric therapy. A study conducted on patients with osteoporosis to determine their vitamin K levels in blood has demonstrated that the vitamin K levels average about one half of those of healthy persons. As a prospective medicine for the treatment of osteoporosis, therefore, the synthetic vitamin K is now under a clinical test. It has been established by the clinical test that in the treatment and prevention of osteoporosis, unlike the case of treating and preventing hemorrhage, the administration of vitamin K in such a large daily dose of not less than 45 mg is effective in increasing the amount of bone. The osteoporosis favors the prevention thereof rather than the treatment to be effected after the onset of the disease. For the sake of this prevention, daily ingestion of the vitamin K from food is commendable. The ingestion of the vitamin K in the dose mentioned above from the existing food seems to be difficult, however, because the question how much of the vitamin K ought to be ingested daily to increase the amount of bone and attain the prevention of osteoporosis remains yet to be elucidated.
The ingestion of vitamin K is preferred to be attained by the daily food as described above. In fact, vitamin K
1
can be taken from greenish yellow vegetables and marine plants and vitamin K
2
from such fermented food products as fermented soybeans (natto). An effort to attain the ingestion of 45 mg of the vitamin K, i.e. an amount reported to be effective in improving osteoporosis, from the commercially available food product, however, actually turns out to be a very difficult task as aptly evinced by the fact that a food product containing 1 ppm of vitamin K, for example, ought to be consumed in such a large amount as 45 kg daily to fulfill the ingestion under discussion. It is natto, among other food products, that has the largest vitamin K content on the order of ten-odd ppm. Even the natto so renowned ought to be consumed in such a large amount as some hundreds of g to several kg daily to fulfill the ingestion. From the standpoint of taste, it is difficult for any person to form the habit of eating the natto in such a large amount as mentioned above daily. In addition to this difficulty, the ingested vitamin K has a short half-life period. The vitamin K has such unsolved problems as failing to manifest the effect thereof fully satisfactory when it is orally ingested independently and tending to entail adverse reactions when it is ingested in an unduly large amount all at once. While the ingestion of the vitamin K in the concentrated form is commendable, the commercially available natural vitamin K concentrate which additionally incorporates therein prepared milk powder for the sake of preventing hemorrhage is expensive and the synthetic vitamin K offered as a pharmaceutical preparation is not usable as food.
Incidentally, in the class of vitamin K's, it are only the vitamin K
1
and K
2
groups that occur in nature. The vitamin K
1
is copiously contained particularly in green vegetables, vegetable oils, and marine products among other kinds of food. Seaweed, laver, and tea leaves, for example, contain some tens of ppm of vitamin K
1
and soybean oil, spinach, and broccoli, for example, contain several ppm of vitamin K
1
. This vitamin K
1
is synthesized by condensing 2-methyl-1,4-naphthoquinone and phytyl acetate. Further, the vitamin K
2
group is known as varying homologs of menaquinone-1 to -14 (MK-1 to MK-14), depending on the difference in chain length. Among other homologs, particularly the menaquinone-7 (occasionally referred to simply as “MK-7” in the present specification) is a typical substance of the vitamin K
2
and is synthesized mainly by
Bacillus subtilis
natto. In nature, the MK-7 is isolated only with unusual difficulty because it occurs in a relatively minute amount in the range of several to ten-odd ppm even in natto and has a short half-life period. So far, the invention of JP-A-08-73,396 has been known as the sole case of succeeding in preparing a lipid having a high MK-7 content.
Thus, quantity production of vitamin K
2
by the use of such a microorganism as
Bacillus subtilis
natto has been attempted. Many studies have been known to have perfected methods for producing natural vitamin K
2
. Methods for collecting vitamin K
2
from the culture broth of a microorganism belonging to genus Flavobacterium (JP-B-07-28,748 and JP-B-07-51,070) and methods for producing vitamin K by inoculating
Bacillus subtilis
natto to soy beam soup stock or soy-bean cake lees and fermenting the microorganism in the medium (JP-A-10-295,393, JP-A-08-19,378, JP-A-08-9,916, and JP-A-08-173,078) may be cited as examples of the outcomes of such studies. In addition to these methods, a method for obtaining a concentrated lipid containing natural vitamin K
2
, particularly natural MK-7, in a large amount by subjecting the fermented cells of
Bacillus subtilis
natto to extraction with such an organic solvent as alcohol, ether, ester, or ketone has been proposed (JP-A-08-73,396). The methods which use such a vitamin K-producing microorganism as Flavobacterium have
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