JPH1056959A - Vitamin k supplementary food and food for preventing or treating osteoporosis - Google Patents

Abstract

PROBLEM TO BE SOLVED: To prepare a vitamin K supplementary food containing vitamin K at a high concentration and a food for preventing or treating osteoporosis capable of preventing or treating the osteoporosis. SOLUTION: This vitamin K supplementary food comprises a bacterium, containing vitamin K at a high concentration and belonging to the genus Lactococcus or the genus Leuconostoc. The bacterium belonging to the genus Lactococcus is Lactococcus lactis ssp. lactis or Lactococcus lactis ssp. cremoris or the bacterium belonging to the genus Leuconostoc is Leuconostoc lactis. The food for preventing or treating osteoporosis comprises the vitamin K supplementary food.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a vitamin K supplement containing a specific lactic acid bacterium containing a high concentration of vitamin K and a food for preventing and treating osteoporosis comprising the vitamin K supplement. It is. The present invention provides a vitamin K supplement food comprising a fermented food containing a specific lactic acid bacterium which is excellent in palatability and which can supplement vitamin K useful for preventing or treating osteoporosis.

[0002]

BACKGROUND OF THE INVENTION Vitamin K was discovered by Dam in Denmark in 1929 as an anti-bleeding factor in chickens. Vitamin K has a common basic skeleton, 2-methyl-
It has a 1,4-naphthoquinone ring, but is divided into several types depending on the side chain at the 3-position. That is, vitamin K found in nature is derived from plant-derived vitamin K ₁ (phyloquinone, P
hylloquinone, MW = 450.68) and vitamin K ₂ derived from general microorganisms (Menaquinone, Menaquinone, MK2-14 depending on the number of prenyl groups in the side chain). There is also a synthetic vitamin K ₃ (menadione).

[0003] The action of vitamin K destabilizes the γ hydrogen of the glutamate substrate, making it more susceptible to CO ₂ attack,
That is, a carboxyl group is introduced at the γ-position (to form Gla). Specifically, osteocalcin (a protein serving as a crystal nucleus of bone matrix vesicles), osteonectin, osteopontin, etc., are formed into a gl form. In addition, suppression of production of bone resorption promoting substances (PGE2, IL-1α, PTH), mononuclear Inhibition of cell differentiation into osteoclasts (Akiyoshi Uchiyama,
CLINICAL CALCIUM, 3, No. 10, p81-86, 1993).

[0004] Vitamin K that is consumed by humans is mainly phyloquinone, while menaquinone is supplied by ingesting natto in animal foods and in Japan. It is also said that phylloquinone is converted into menaquinone-4 (MK4) in the body (Billeter M. & Martius C., Biochemische Z.
eitschrift, 333, p430-9, 1960).

On the other hand, there are many reports that microorganisms in the intestinal flora produce menaquinones (at the cell membrane), and this is a source of vitamin K for humans. However, papers that have proposed this theory using animals often fail to solve the problem of animal feces, or overlook that the antibiotics used are vitamin K inhibitors, and they are still missing. No confirmation has been obtained. It may also be difficult to think of taking up a significant amount of fat-soluble substances in an environment where the large intestine is free of bile acids (Ichihashi et al., J. Nutr., 122, p506-12, 19
92). The only compelling report is that 90% of menaquinone in a human liver was MK7-13, which was similar in composition to the intestinal flora of that person (Conly
JM, & Stein K., Am. J. Gastroenterol., 87, p311
-6, 1992).

[0006] However, the fact that a certain amount of menaquinone is supplied from the intestinal flora, if any, is only a part of the required amount. It is said that banning foods containing high phylloquinone leads to vitamin K deficiency. et al., (A
m. J. Clin. Nutr., 47, p475-80, 1988). It can be said that the supply of vitamin K from the intestinal flora is not sufficient (Bentley R., & Meganathan R., Microbi
ol. Rev., 46, p241-80, 1982).

[0007] As a required amount of vitamin K, it is said that supplementation of 1.0 µg / kg is necessary to maintain human prothrombin concentration. In Germany and the United States, the required amount is set at 65-80 μg / day (almost 1 μg / kg). On the other hand, in Russia, it is as high as 200 to 300 μg. The intake of adolescents in the United States is about 100 μg / day (Sutt
ie JW, et al., Am. J. Clin. Nutr., 47, p475-80,
1988).

[0008] Then, it is supposed that vitamin K is not deficient. Actually, there are some reports that the serum vitamin K concentration is lower in osteoporosis patients, especially in fractured patients than in non-fractured patients. (Hart JP, et a
l., J. Clin. Endocr. Metab., 60, p1268-9, 1985;
Masataka Shiraki, Seminar on Vitamin K Function (Proceedings), 5, p97-109, 1990; Kanagi et al., Journal of the Japan Society for Bone Metabolism, 1
2, No. 2, p242, 1994). In the case of renal dialysis patients, 0.8
A fracture has occurred following the nmole / L serum (newspaper article; Assistant Professor Kohlmeier of the University of North Carolina, 1995).

In addition, the causes of vitamin K deficiency in breastfeeding infants are that there are many bifidobacteria which do not have menaquinone synthesizing ability, the hepatic accumulation of menaquinone derived from intestinal bacteria is slow, and the mothers lack vitamin K intake. Therefore, it is considered that the insufficient transfer to breast milk is a major factor. Because intramuscular injection of vitamin K to prevent neonatal melena may increase the risk of developing childhood cancer (J. Golding, et a
l., BMJ, 305, p341-6, 1992), it is also recommended that nursing women take sufficient vitamin K to increase the content in breast milk.

In view of the above, it is considered that there is no remarkable deficiency of vitamin K because the sufficiency of vitamin K is conventionally measured by the prothrombin time lacking in sensitivity. Further investigation is required to determine whether the sufficiency of vitamin K, which has many physiological functions including unknown ones, is properly evaluated (Latest Nutrition, 6th edition, p202, Kenzosha, 1991).

[0011] On the other hand, in a study in which vitamin K is administered to humans to examine the effect on bone formation, there is a study in which vitamin K is administered to an elderly woman with osteoporosis to observe an increase in bone mass (Hajime Orimo, et al.). , Clin. Eval., 20, p45-100, 1992; 45 mg /
Administration of MK-4 for more than one day increases the bone mass of the radius after half a year).
In addition, Knappen et al. Reported that administration of 1 mg of phylloquinone to postmenopausal women with high Ca looseness for 2 weeks reduced urinary Ca excretion, increased urinary hydroxyproline, and made osteocalcin into Gla (Knapen MH, et al.,
Annals of Intern Med, 111, p1001-1005, 1989). On the other hand, toxicity due to large dose administration of filoquinone or menaquinone is not known.

[0012]

As described above, vitamin K is an important vitamin for preventing or treating osteoporosis, and foods containing it include spinach, turnip,
Green vegetables such as broccoli were known. further,
As a fermented food rich in vitamin K, there is natto using natto bacteria unique to Japan. Natto is thought to be related to fewer fractures in the Japanese, especially in eastern Japan, which consumes a lot of natto (Tokyo Shimbun, Takayuki Hosoi, Director of the University of Tokyo, March 7, 1996). The so-called Japanese paradox, which says that Japanese women who have 10-20% less bone mass than Western Caucasians who take a lot of calcium and have only half the fractures, says that there is a reason for traditional Japanese food and natto that consume a lot of vegetables and seaweed. Have been done.

However, in modern Japanese eating habits in which refined and processed foods are flooding, or in the case of unbalanced eating habits in which foods such as green vegetables and natto of vitamin K cannot be sufficiently taken, a vitamin K deficiency occurs. Could be. Also, the vitamin K content in animal foods is not so high,
A bias toward meat eating can lead to a vitamin K deficiency (paradox described above). In fact, as mentioned above, vitamin K is clearly deficient in elderly people and osteoporosis, particularly in fractured people, people who eat unbalanced foods, those who take antibiotics, and pregnant and nursing women.

For the above reasons, it seems that vitamin K needs to be replenished to an extent not too much. It is considered appropriate that the amount is 10 to several tens μg per day. However, 10 to several tens μg of vitamin K per day
In order to consume, 100g of green vegetables must be consumed.

There have been many reports that microorganisms are required to supply vitamin K. However, most of which was placed a preparation of vitamin K ₂ or K ₃ pharmaceutical use in mind, in many cases bacteria not suitable for food are used. Menaquinone (extract, mainly composed of menaquinone-4) as a food additive other than chemically synthesized products is included in the list of existing additives (Notification No. 166, 416 of the Ministry of Health and Welfare on August 10, 1995) Has been
For this purpose, the genus Arthrobacter, a kind of acetic acid bacterium, is used (JP-A-63-23092). Vitamin K
Although it is good to purify and use acetic acid, there are many disadvantages in using acetic acid bacteria or fermentation products together.

As edible microorganisms, lactic acid bacteria and yeast seem to be the most promising, but the former is, for example, bifidobacterium does not produce vitamin K and Bifidobacterium bi
Menadione can be a growth-promoting factor, such as fidum var.
(Glick MC, et al., J. Bacteriol., 77, p230-6,19
59). Most lactococci and bacilli have poor ability to produce vitamin K. Lactobacillus casei subsp. Rhamnosus AT
CC7469 contains 60 nmole of vitamin K per gram of bacterial protein
Reported that the produce (corresponding to a dry cell g per about _{6μg) (Hess A. Etal.} , J. General Microbiol., 115, p
247-52, 1979), even though the presence of vitamin K is qualitatively indicated (Collins MD & Jones D., Microbiol. R
ev., 45, p316-54, 1981), none of which quantitatively shows the vitamin K content of lactic acid bacteria. On the other hand, yeast almost always produces ubiquinone and is not a source of vitamin K. In addition, there has been no report on the use of a specific lactic acid bacterium containing a high concentration of vitamin K in a vitamin K supplement.

[0017]

[Means for Solving the Problems] Under such circumstances,
In view of the above-mentioned prior art, the present inventors have investigated edible fermentation microorganisms, particularly Bifidobacterium, Lactobacillus, Streptococcus, Lactococcus, Leuconostoc, in order to search for a new supply source of vitamin K. We conducted exploratory research mainly on lactic acid bacteria such as genera. As a result, the present inventors have found that a certain group of lactic acid bacteria has a potential as a vitamin K supplement source, contrary to what is expected from the conventional knowledge, and have completed the present invention.

That is, the present inventors have conducted intensive studies with the aim of developing an edible fermentation microorganism useful as a new supplemental source of vitamin K and a vitamin K supplement containing the edible fermentation microorganism. As a result, Lactococcus lactis ssp.cremoris and Lactococcus
It was found that lactis ssp. lactis, Leuconostoc lactis and the like produce several tens to two hundred and several tens of micrograms of menaquinone per gram of freeze-dried cells (Table 1). According to studies conducted by the present inventors, this value is, for example, the aforementioned Lactobac
illus casei subsp. rhamnosus ATCC7469 compared to 3
Since the amount reaches about 43 times, it is found that the content is in a range sufficiently practical as a vitamin K supplement source.

According to the present invention, a lactic acid bacterium producing high vitamin K can be cultured and the cells can be fed. In the present invention, the form of the cells may be either live cells or dead cells, and the type and form of the vitamin K supplement are not particularly limited, but tablet confections, various powdered foods, and beverages And oil and fat foods are exemplified as suitable ones. The most economical and easy-to-ingest form is to produce fermented milk and lactic acid bacteria beverages using lactic acid bacteria that produce high levels of vitamin K. In the former case, the vitamin K supplement food is produced by incorporating a bacterium belonging to the genus Lactococcus or a bacterium belonging to the genus Leuconostoc containing a high concentration of vitamin K into the food by an appropriate method. As a specific method of containing the above bacteria,
Although not particularly limited, for example, freeze-dried cells, spray-dried cells, wet cells, or a method of adding a crushed product or a fat-soluble fraction thereof to a food material, mixing with a powdered food Method, and the like. In the latter case,
For example, for raw materials such as skim milk powder, yeast extract, L-cysteine hydrochloride, calcium carbonate, and sugar solution, a bacterium belonging to the genus Lactococcus or a bacterium belonging to the genus Leuconostoc is used as a starter and fermented according to a conventional method. Fermented milk and the like are produced. Similarly, a fermented product can be diluted with a syrup solution to produce a fermented bacteria beverage.In addition, conventionally, fermented milk and the like have been produced using lactic acid bacteria belonging to the genus Lactobacillus. A high concentration, for example, vitamin K is added to 20 g of freeze-dried cells
μg or more, an example of producing a food containing a bacterium belonging to the genus Lactococcus or a genus Leuconostoc containing, or a fermented food containing a high concentration of vitamin K using the bacterium as a starter No examples have been reported so far. Further, based on the present invention, a plant extract, for example, soybean milk, almond milk, rice milk, fruit juice, etc., can be fermented with lactic acid bacteria producing high amounts of vitamin K to produce and ingest fermented foods.

[0020]

According to the present invention, a vitamin K supplement containing a bacterium belonging to the genus Lactococcus or Leuconostoc containing a high concentration of vitamin K, and a food for preventing or treating osteoporosis comprising the vitamin K supplement. Can be provided. Menaquinone produced by the genus Lactococcus has a side chain of 6 to 9 (MK6 to 9) and the main component is MK9 (Collins MD & Jones D., Microbiol.
Rev., 45, p316-54, 1981), which is converted to MK4 in vivo and used (Masaki Kato et al., 50th Annual Meeting of the Japanese Society of Nutrition and Food Science, p152, 1996). The supplemented vitamin K normalizes the serum vitamin K concentration, and can be expected to have an effect of preventing and treating diseases related to bone formation.

[0021]

Next, the present invention will be described with reference to examples.
The present invention is not limited to the embodiment. Example 1 A lactic acid bacterium of the genus Bifidobacterium, Lactobacillus, Streptococcus, Lactococcus, Leuconostoc, etc. was placed in a compatible liquid medium (modified Rogo).
(sa medium), and cells were collected and washed. Vitamin K was extracted from the wet cells and qualitatively and quantitatively analyzed by thin-layer chromatography. The results are shown in Table 1.

[0022]

[Table 1]

As shown in Table 1, it was found that Lactococcus and Leuconostoc spp. Have high menaquinone contents.

Example 2 The genus Lactococcus and Leuconostoc were searched in more detail. Various bacterial cells are cultured in each compatible liquid medium (modified Rogosa medium), collected, washed, and vitamin K is extracted from the lyophilized cells and qualitatively and quantitatively analyzed by thin-layer chromatography. Was. The results are shown in Table 2.

Vitamin K supplementation should be 10 to several tens of μm a day.
g is a guide, so if this is normal fermented milk 200mL
It can be inferred that the strains underlined in Table 2 are preferable as practical vitamin K-producing lactic acid bacteria. As shown in Table 2 below, among others, Lactoc
occus lactis ssp.lactis YIT2016 (ATCC 13675), ss
p.cremoris YIT2007 (ATCC 19257), ssp.cremoris Y
IT2011 (FERM P-15769), ssp.cremoris YIT2012 (FERM
P-15770), Leuconostoc lactis YIT3001 (ATCC 19256)
Was found to be preferable.

[0026]

[Table 2]

EXAMPLE 3 200 containing skim milk powder (20 g), yeast extract (0.2 g), L-cysteine hydrochloride (0.06 g) and calcium carbonate (0.4 g)
Make a solution of mL and sterilize at 121 ° C for 15 minutes.
1% of starter culture of occus lactis ssp. cremoris YIT2011 and YIT2012 was inoculated. Further, a similar milk powder solution was prepared by further increasing the yeast extract to 1 g.
After incubating for 15 minutes with, a 2% starter medium of Leuconostoc lactis YIT3001 was inoculated and cultured at 30 ° C until the milk solidified in an incubator to produce fermented milk. The fermented milk obtained was used as a lyophilized powder, and the fat-soluble fraction was extracted with a mixed solution of chloroform and methanol, and the amount of menaquinone was determined by HPLC. As a result, YIT2011 strain fermented milk 200m
10.8 μg of menaquinone was obtained in terms of L, 5.2 μg of YIT2012 and 13.8 μg of menaquinone of YIT3001. In addition, the same treatment and analysis were performed on a medium not inoculated with the starter used as a control, and as a result, menaquinone was not detected.

Example 4 200 containing 4 g of lactose and 0.4 g of calcium carbonate
mL of soymilk was autoclaved at 115 ° C for 15 minutes, and La
cremoris YIT2011 starter was inoculated with 1% of ctococcus lactis ssp. At this time, the acidity was 5.7 and the pH was 4.92. The fermented curd was homogenized, and syrup was mixed to produce fermented soymilk. The liquid fermented soy milk was also functionally good. A part of the homogenized curd was used as a lyophilized powder, and the fat-soluble fraction was extracted with a mixed solution of chloroform and methanol, and menaquinone was quantified by HPLC. As a result, in YIT2011 strain, 12.8 μg of menaquinone was detected in terms of 200 mL of fermented milk.

[0029]

As described above in detail, the present invention relates to a vitamin K supplement containing a bacterium belonging to the genus Lactococcus or a bacterium belonging to the genus Leuconostoc containing a high concentration of vitamin K. Therefore, according to the present invention, a vitamin K supplement containing high levels of vitamin K can be provided. The vitamin K supplement food of the present invention is useful as an osteoporosis prevention / treatment food for preventing or treating osteoporosis. According to the present invention, vitamin K-containing fermented milk and lactic acid bacteria beverages can be easily produced, so that the selection range of vitamin K supplements can be further expanded than before.

──────────────────────────────────────────────────の Continuation of the front page (51) Int.Cl. ⁶ Identification number Agency reference number FI Technical display A61K 35/74 ABJ A61K 35/74 ABJA (72) Inventor Natsuko Tamura 1-chome, Higashi-Shimbashi, Minato-ku, Tokyo No. 1-19 Yakult Honsha

Claims (6)

[Claims] 1. A vitamin K supplement comprising a bacterium belonging to the genus Lactococcus or a bacterium belonging to the genus Leuconostoc, which contains a high concentration of vitamin K. 2. The vitamin K supplement according to claim 1, wherein the vitamin K is vitamin K ₂ (menaquinone). 3. A bacterium belonging to the genus Lactococcus or a bacterium belonging to the genus Leuconostoc, which contains vitamin K at a high concentration, converts vitamin K to 2 g per lyophilized bacterial cell.
The vitamin K supplement according to claim 1 or 2, which is a bacterium containing 0 μg or more. 4. The bacterium belonging to the genus Lactococcus,
The vitamin according to claim 1, 2 or 3, wherein the bacterium is Lactococcus lactis ssp. Lactis or Lactococcus lactis ssp. Cremoris or the bacterium belonging to the genus Leuconostoc is Leuconostoc lactis. K supplements. 5. The vitamin K supplement according to claim 1, wherein the food is fermented milk. 6. A food for preventing and treating osteoporosis, comprising the vitamin K supplement according to claim 1.