JPH09194384A - Mineral absorption accelerating agent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a mineral absorption accelerating agent containing cells of a bacterium belonging to the genus Bifidobacterium as an active ingredient, having an accelerating action on the increase of a bone strength and a bone mineral content, and useful for the prevention and treatment of osteoporosis. SOLUTION: This mineral absorption accelerating agent contains cells of a bacterium belonging to the genus Bifidobacterium such as Bifidobacterium longum, B. bifidum, B. adolescentis, B. breve and B. infantis, as an active ingredient and e.g. by 1×10<4> to 1×10<12> /g number of cells as live cells. These cells are obtained by inoculating the prescribed cells to a liquid medium for propagation such as Brigg's liver broth, culturing under an anaerobic condition, at 37 deg.C for 16hr, then collecting cells by a centrifugal separation, washing and lyophilizing. Further, preferably this medicine is taken by >=1×10<8> cells daily in the case of an adult.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a mineral absorption enhancer containing a bacterium belonging to the genus Bifidobacterium as an active ingredient. The mineral absorption promoter of the present invention is useful for the prevention and treatment of osteoporosis and the like.

[0002]

2. Description of the Related Art In recent years, Japan has been known as one of the longest-lived countries in the world, but on the other hand, osteoporosis, which is found in the elderly and postmenopausal women, has become a serious problem.
Osteoporosis is thought to be caused by a decrease in calcium absorption capacity with age, a decrease in the secretion of active vitamin D ₃ , a hormone imbalance, etc. However, it is often associated with bone fractures, etc. It is thought that it will become more and more serious illness in the future because it requires physical activity and physical activity is extremely limited. In addition, not only the elderly but also the increased fracture rate of children has become a situation that cannot be ignored. It is considered that the fractures of young people are caused by insufficient intake of calcium and insufficient bone metabolism due to unbalanced diet and lack of exercise, while having excessive energy. Therefore, bone-related illness is now a problem common to Japanese people regardless of age.

Under these circumstances, studies have been conducted on substances that improve calcium supplementation and absorption. Lactose is the most well-known substance that promotes the absorption of calcium (Takero Goto, Hiromaro Seki, Nutrition and Foods, vol.17, p.188, 1964). In addition, lactulose, sorbitol, etc. are known to have a calcium absorption promoting effect (Kazuharu Suzuki et al., Eishoku magazine, vol.38, p.3).
9, 1985). Further, casein phosphopeptide, which is a partial degradation product of casein, is known to prevent insolubilization of calcium and enhance the absorption efficiency of calcium (JP-A-58-170440 and JP-A-5-336894). ). Further, it is also known that oligosaccharides, which are growth factors of Bifidobacterium, have a calcium absorption promoting effect (JP-A-4-134031, JP-A-4-349868,
JP-A-7-67575). However, although these substances contribute to the supplementation of calcium and the improvement of absorption, they do not always directly act on the deposition of bone mineral and the increase of bone strength.

On the other hand, Bifidobacterium has been studied for a long time as a useful bacterium in humans, and has an intestinal regulating action, a blood lipid improving action, a growth inhibiting action against harmful bacteria such as putrefactive bacteria, and the like. Are known. As a result, it has been suggested that it is useful for improving various organ disorders, carcinogenesis, arteriosclerosis, hypertension, liver disorders, reduction of autoimmune ability, etc. Abstracts, p.11, 1995). Further, by assimilating a fermentation substrate such as oligosaccharide with a fermenting bacterium such as Bifidobacterium, acetic acid produced by fermenting in the intestine,
It has been reported that organic acids such as lactic acid, propionic acid and butyric acid enhance the solubility of calcium in the intestine and enhance the absorption rate of calcium (Ohta, A. et al., J. Jpn. Soc. Nu.
tr. FoodSci., vol.46, p.123, 1993, Chonan, O. et a
l., J. Nutr. Sci. Vitaminol., vol.41, p.95, 1995)
. However, it has not been known at all that the Bifidobacterium bacterium itself acts on the host organism, affects hormone metabolism and bone metabolism, and promotes bone mineral deposition and bone strengthening. It was

[0005]

DISCLOSURE OF THE INVENTION The present inventors have been conducting research on effective use of Bifidobacterium and at the same time conducting research on mineral absorption promoters having high preventive and therapeutic effects on osteoporosis. It was found that ingestion of Bifidobacterium cells can promote bone mineral deposition and bone strengthening, and completed the present invention. As will be described later, this effect is not due to the organic acids produced by conventional fermenting bacteria but due to the action of the bacterium of Bifidobacterium itself. Therefore, the present invention is to provide a mineral absorption enhancer containing a Bifidobacterium bacterium as an active ingredient.

[0006]

The mineral absorption promoter of the present invention is characterized in that it contains cells of the genus Bifidobacterium as an active ingredient. The Bifidobacterium usable in the present invention, Bifidobacterium Russia <br/> gum (Bifidobacterium longum), Bifidobacterium bifidum (Bifidobacterium bifidum), Bifidobacterium address Sen infantis ( Bifidobacterium
adolescentis ), Bifidobacterium breve (B
ifidobacterium breve) and it can be exemplified Bifidobacterium infantis (Bifidobacteriuminfantis).

As a method for preparing these Bifidobacterium genus cells, for example, Brigg's Liver Broth, which is used for usual culture of Bifidobacterium genus
Inoculate the growth liquid medium such as
After culturing for 16 hours, the resulting culture was centrifuged (10,000
Xg, 15 minutes) to collect the cells, and the cells may be washed appropriately and freeze-dried. The powder cells of the Bifidobacterium genus are adjusted so that the number of bacteria in the mineral absorption promoter is 1 × 10 ⁴ to 1 × 10 ¹² / g.
It is also possible to culture the cells of the genus Bifidobacterium in a medium such as milk or skim milk and use the culture as it is as a mineral absorption promoter.

[0008]

DETAILED DESCRIPTION OF THE INVENTION The present invention provides Bifidobacterium longum (Bifidobacterium longum), Bifidobacterium bifidum (Bifidobacterium bifidum), Bifidobacterium address Sen infantis (Bifidobacte
rium adolescentis ), Bifidobacterium breve , Bifidobacterium
It is a mineral absorption enhancer containing, as an active ingredient, cells of a genus Bifidobacterium such as Bifidobacterium infantis . And the number of bacteria of this bacterium is 1 × 10
It may be in any form such as food and drink, or a preparation as long as it is adjusted to ⁴ to 1 × 10 ¹² / g, preferably 1 × 10 ⁶ to 1 × 10 ¹¹ / g . For example, the cultured bacterial cells may be freeze-dried, and about 3 g of the resulting bacterial cell powder in a degassed package may be ingested as it is. You may ingest the thing of the capsule form prepared according to Kaihei 1-228456 publication.
Ordinary fermented milk may be inoculated with Bifidobacterium spp. And ingested in the form of hard-type or drink-type fermented milk.

Further, the mineral absorption promoter of the present invention comprises:
Bifidobacterium genus growth factors such as oligosaccharides and highly absorbable calcium agents may be used in combination with these cells.

The intake amount of the mineral absorption enhancer of the present invention is
Adults should take Bifidobacterium cells 1 / day
× 10 ⁸ or more, i.e., 1 × 10 ⁷ / g of powder bacteria a long if one day 10g or more, it is preferable to ingest daily 100g or more if fermented milk 1 × 10 ⁶ / g.

Next, the present invention will be described in detail by showing Examples and Test Examples.

Example 1 Brigg's Liver Broth having the composition shown in Table 1
Sterilize 10 liters at 121 ° C for 15 minutes, then add to this medium:
Bifidobacterium longum SBT-2928 (FERM P-1065) pre-cultured in the same medium
2% of the culture solution of 7) was inoculated and cultured at 37 ° C. for 16 hours while maintaining the pH of the medium at 6.5 to 6.8 with 1N ammonia water under anaerobic conditions with aeration of carbon dioxide.

[0012]

[Table 1] ───────────────────── Tomato juice 400 ml Neopeptone (manufactured by Difco) 15 g Yeast extract (manufactured by Difco) 6 g Liver extract 70.6 ml Glucose 20 g Soluble starch 0.5 g Sodium chloride 5 g Tween80 1 g L-Cysteine hydrochloride 0.2 g Water (pH 6.8) 525 ml ──────────────────── ──

After the culture is completed, the obtained culture is centrifuged.
(10,000 × g, 15 minutes) to collect the bacterial cells, which were washed 3 times with physiological saline, and then added with an equal amount (wet weight) of 1% sodium glutamate to reduce the concentration to 10%. It was dispersed in skim milk and freeze-dried. Thus, 70 g of powdered cells of Bifidobacterium longum was obtained. In addition, the number of bacteria of this powdered bacterium is 1 × 10
It was ¹⁰ / g.

[0014]

Example 2 1 liter of the raw material having the composition shown in Table 2 was used.
After sterilizing at 10 ℃ for 10 minutes, Bifidobacterium breve (Bifidobacte
riumbreve) Inoculate 2% of ATCC-15700 culture solution at 37 ℃
After culturing for 16 hours, 1.1 kg of fermented milk was obtained. The number of bacteria in this fermented milk was 1 × 10 ⁷ / ml.

[0015]

[Table 2] ──────────────── Skim milk powder 10 (wt%) Yeast extract 0.5 Water 89.5 ─────────────────

[0016]

Example 3 Under the same conditions as in Example 1, Bifidobacterium bifidum ATCC-
29521, Bifidobacterium addressacetis (B
ifidobacterium adolescentis) ATCC-15703 and Bifidobacterium infantis (Bifidobacterium
infantis ) ATCC-15697 was cultivated,
70g was obtained.

[0017]

[Test Example 1] As a test animal, a Wistar aseptic rat having an initial body weight of 80 to 90 g was used for an animal experiment. That is, as a group of the present invention, powdered cells of Bifidobacterium longum obtained in Example 1 were suspended in sterile physiological saline so that the number of cells was 1 × 10 ⁸ / ml, and 5 One rat was orally administered with 1 ml of cell suspension every day for 4 weeks. On the other hand, as a control group, Bacteroides obtus ( Bacteroides obtus) prepared by the same method as in Example 1
teroides ovatus ) SBT3175 powdered cells with a cell count of 1 x 10
^The suspension was suspended in sterile physiological saline at ⁸ / ml, and 5 rats were orally administered with 1 ml of cell suspension for 4 weeks by an oral probe. Also, the feed is a commercially available radiation-sterilized solid feed (NM
F feed, manufactured by Oriental Yeast Co., Ltd.) was freely taken.
After 4 weeks, the femur was removed under ether anesthesia, and the bone breaking strength was measured with a rheometer (RX-100, manufactured by Aitechno Co., Ltd.) to obtain the bone strength. The result is shown in FIG. Blood was collected from the posterior vena cava to separate serum, and the parathyroid hormone and calcitonin concentrations in the serum were measured. The results are shown in FIGS.

The bone strength of the group of the present invention was significantly higher than that of the control group, and it was found that the cells of the genus Bifidobacterium have a bone strengthening effect. In addition, the parathyroid hormone concentration in serum was significantly higher in the group of the present invention than in the control group, and it was confirmed that production of active vitamin D was promoted and calcium absorption was enhanced. Therefore, it is understood that ingestion of Bifidobacterium bacterium can affect the metabolism of the living body and promote the absorption of minerals. In addition, no difference was observed in serum calcitonin concentration, and no effect on bone resorption was observed.

[0019]

[Test Example 2] As a test animal, a Wistar aseptic rat having an initial body weight of 80 to 90 g was used for an animal experiment. That is, as a group of the present invention, fermented milk containing the Bifidobacterium breve cells obtained in Example 2 was administered to 5 rats with an oral sonde every day at 1 ml.
The cell suspension of was administered for 4 weeks. On the other hand, as a control group,
Powdered bacterial cells of Bacteroides ovatus SBT3175 prepared in the same manner as in Example 1 were suspended in sterilized skim milk to a bacterial count of 1 × 10 ⁷ / ml, and 5 Rats were orally administered with 1 ml of cell suspension every day for 4 weeks. In addition, a feed having the composition shown in Table 3 was prepared and 50 kG
After being sterilized with radiation of y, the diet was given ad libitum.
Note that galactooligosaccharide was added to the feed in order to see the effect of mineral absorption by the fermentation products of intestinal bacteria known in the past.

[0020]

[Table 3] ────────────────────── Casein 20.0000 (wt%) L-Cysteine 0.3000 Corn starch 39.7486 α-corn starch 13.2000 Sucrose 5.0000 Galactooligosaccharide 5.0000 Soybean oil 7.0000 Cellulose 5.0000 Choline bitartrate 0.2500 Butylhydroquinone 0.0014 Vitamin mixture ^* 1.0000 Mineral mixture ^* 3.5000 ────────────────────── ^* AIN-93G (J. Nutr. 123) , 1939-1951 (1993)).

Then, after 4 weeks, the femur was removed under ether anesthesia, and the femur was extracted by a conventional method (ICP emission analysis, Kei Fuwa, edited,
p.196 (1980) issued by Nankodo)
The amount of bone mineral was measured with an optical emission spectrometer. In order to examine the relationship with the fermentation products, the cecum was removed, the pH was measured directly with a pH meter, and then the organic acid content was measured with a carboxylic acid analyzer. The results are shown in Table 4.

[0022]

[Table 4] ───────────────────────────────── Inventive group Control group ──────── ───────────────────────── Calcium 96.1 ± 11.8 ^* (mg / bone) 85.2 ± 5.2 (mg / bone) Phosphorus 47.8 ± 6.1 ^* 41.6 ± 2.4 Magnesium 2.7 ± 0.3 ^* 2.5 ± 0.1 ───────────────────────────────── pH 6.9 ± 0.2 6.8 ± 0.2 Lactic acid 1.4 ± 0.3 (mg / cecum) 1.0 ± 0.1 (mg / cecum) acetic acid 3.1 ± 1.9 ^* 8.8 ± 1.6 propionic acid not detected 2.9 ± 0.3 ─────────────────── ─────────────── ^* Significantly different from the control group (p <0.05)

The amount of bone mineral in the group of the present invention was significantly higher than that in the control group. Therefore, it is understood that the bone mineral amount can be increased by ingesting the cells of Bifidobacterium. In addition, the amount of organic acids in the control group was significantly larger than that of the present invention group, and the mineral absorption promoting action by the ingestion of Bifidobacterium was not the action by the organic acids through fermentation of enterobacteria. It was confirmed.

[0024]

[Test Example 3] BALB / c aseptic mice having an initial body weight of 25 to 30 g were used as test animals, and animal experiments were conducted. That is, the present invention group, a bicycloalkyl obtained in Example 3 Bifidobacterium bifidum (Bifidobacterium bifidum), Bifidobacterium address Sen infantis (Bifidobacterium
adolescentis ) and Bifidobacterium infantis powder cells were suspended in sterilized physiological saline so that the number of cells was 1 × 10 ⁷ / ml, and 5 mice were treated with an oral probe for 0.3 days each day. A bacterial cell suspension of ml was administered for 4 weeks. On the other hand, as a control group, Bacteroides fragilis, which was prepared in the same manner as in Example 1 (Bacteroides
fragills ) ATCC-25285 and Eubacterium Lentum ATCC-25559 powdered cells were suspended in sterile physiological saline so that the number of cells was 1 × 10 ⁷ / ml, and 5 Each mouse was orally administered with 0.3 ml of cell suspension every day for 4 weeks. In addition, a sterile group to which no bacterial solution was administered was also added to the experimental group. As the feed, a commercially available radiation-sterilized solid feed (NMF feed, manufactured by Oriental Yeast Co., Ltd.) was freely taken. Then, after 4 weeks, the femur was removed under ether anesthesia, and a rheometer (RX-100, manufactured by Aitechno Co., Ltd.)
The bone breaking strength was measured with. The result is shown in FIG. The bone breaking strength was significantly higher in the present invention group than in the control group and the aseptic group, and it was found that the cells of the Bifidobacterium genus have a bone strengthening effect.

[0025]

Example 4 Bifidob Longum
Acterium longum ) SBT-2933R (FERM P-8743) was inoculated into 100 ml of sterilized skim milk similar to that shown in Table 2 at 2%, and at 37 ° C.
After culturing for 16 hours, 108 g of cultured milk was obtained. In addition, Bifidobacterium longum (Bifidobacterium
The number of longum ) was 1 × 10 ⁷ / ml. This cultured milk 7
5 g was used and diluted with 75 g of 16% reduced skim milk to prepare 150 g of bacterial cell fluid. On the other hand, 3% by weight of an emulsifier (tetraglycerin pentastearate) was added to 1 kg of butter oil dissolved by heating and mixed. Then, 150 g of the bacterial cell liquid was sequentially added to 100 g of butter oil kept at 35 ° C. and emulsified by stirring at 1,000 rpm for 3 minutes with a lab disper. Next, after dissolving 4% by weight of calcium lactate and 1% by weight of xanthan gum in 750 g of water at 35 ° C., 250 g of a mixed emulsion of the cell fluid and butter oil prepared above was added and dispersed. This dispersion was dropped into the 0.5 wt% sodium alginate solution from the tip of the nozzle to obtain spherical capsules having a diameter of 1 to 5 mm. The capsule was washed with water and dried to prepare 150 g of a viable bacterial preparation of Bifidobacterium. In addition, Bifidobacterium longum (Bifidobacterium
The number of longum ) was 4 × 10 ⁸ .

[0026]

[Example 5] Yogurt mix 1 containing milk as a main component
After sterilizing kg for 30 minutes at 95 ℃, Bifidobacterium
Longum (Bifidobacterium longum ) SBT-2928 (FERM P
-10657), Streptococcus thermophilus (Str
eptococcus thermophilus ) SBT-1021A (FERM P-1065
8), Lactobacillus acylphus
idophilus) SBT-2056 (FERM P-8744) separately inoculated with 1.5% by weight of separately manufactured bulk starters,
Fermentation was carried out at 39 ° C with a target acidity of 0.80% after fermentation, and after completion of fermentation, the mixture was rapidly cooled to 10 ° C to obtain 1 kg of fermented milk.
The fermentation time was completed in 4 hours, and the number of Bifidobacterium longum bacteria at that time was 1 × 10 ⁸ / g. This fermented milk was used as a mineral absorption promoter.

[0027]

Example 6 Bifidob Longum
Acterium longum ) SBT-2933R (FERM P-8743) was inoculated into 200 liters of the same medium as shown in Table 1 at 2% and incubated at 37 ° C.
After culturing for 16 hours, the bacterial cells were collected and freeze-dried to obtain 1 kg of powdered bacterial cells. The number of bacteria in this powdered bacterium is 1 × 10 ¹¹ / g
Met. Using 800 g of this powdered bacterial cell, cornstarch
200 g, galacto-oligosaccharide 600 g and beef bone powder 400 g were mixed with a kneader, and then 250 ml of water was spray-dried and kneaded.
This mixture was granulated with a single-screw oscillator with a 20-mesh screen set, dried with a fluidized bed drier, pulverized with a flash mill, and sized to produce a tableting powder. Then, this powder and 40 g of sucrose fatty acid ester as a lubricant were mixed in a V-type mixer and tableted with a tablet machine in which a punch having a diameter of 11 mm was set, to obtain tablets 2,3 having an average weight of 0.35 g.
000 grains were produced. The number of bacteria in this tablet is 1
It was × 10 ⁸ / g. Therefore, 7 to 10 tablets of this tablet should be taken daily.

[0028]

INDUSTRIAL APPLICABILITY The mineral absorption enhancer of the present invention contains a bacterium of Bifidobacterium as an active ingredient and exerts an influence on the hormonal metabolism and bone metabolism of a living body to promote an increase in bone strength. It also has the effect of promoting an increase in bone mineral content. Therefore, ingestion of the mineral absorption enhancer of the present invention can contribute to the prevention or treatment of osteoporosis.

[Brief description of the drawings]

FIG. 1 shows the results of bone fracture strength measurement in Test Example 1.

FIG. 2 shows the results of parathyroid hormone measurement in Test Example 1.

FIG. 3 shows the results of calcitonin measurement in Test Example 1.

FIG. 4 shows the results of bone strengthening action in Test Example 3.

Claims (4)

[Claims] 1. A mineral absorption enhancer comprising a Bifidobacterium bacterium as an active ingredient. Wherein cells of Bifidobacterium is Bifidobacterium longum (Bifidobacterium long
um ), Bifidobacte
rium bifidum), Bifidobacterium address Sen Tisdale (Bifidobacterium adolescentis), Bifidobacterium breve (Bifidobacterium breve) and Bifidobacterium infantis (Bifidobacteri
The mineral absorption enhancer according to claim 1, which is at least one kind of bacterial cells selected from the group consisting of um infantis ). 3. The mineral absorption enhancer according to claim 1, wherein the cells of the genus Bifidobacterium are viable cells. 4. The mineral absorption enhancer according to claim 1, which contains 1 × 10 ⁴ to 1 × 10 ¹² / g in terms of the number of Bifidobacterium cells.