JP2000239180A - Calcium crystallization inhibitory absorption facilitative agent containing protein obtained from microbial cell body of bacterium having calcium crystallization inhibitory effect as active ingredient, and ingesta and feed comprising the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a calcium crystallization inhibitory absorption facilitative agent which controls the crystallization of calcium in the body and facilitates the absorption of calcium in the intestine by including a protein obtained from microbial cell bodies of bacteria having calcium crystallization inhibitory effect. SOLUTION: This calcium crystallization inhibitory absorption facilitative agent is obtained by including a protein obtained from microbial cell bodies of bacteria having calcium crystallization inhibitory effect. As the above bacteria, Bacillus species 20-1 (FERM P-17193), Strain 19 (FERM P-17194), Strain 20 (FERM P-17195) and Strain 25W (FERM P-17196) inhabiting Vanda lake in South Pole are particularly illustrated. The above protein can be extracted by a generally known microbial cell body crushing method and a physical crushing method, e.g. pressure type cytoclasis, mechanical grinding, ultrasonication, homogenizer or the like, is preferably used. Further, the ingest amount is preferably 0.1-100 mg/kg/day.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a novel use of a protein obtained from a bacterial cell, and more specifically, a protein obtained from a bacterial cell having a calcium crystallization inhibitory activity as an active ingredient. The present invention relates to a calcium crystallization inhibitor / absorption enhancer, and a food, drink, and feed containing the same.

[0002]

2. Description of the Related Art Calcium is said to be the most abundant of the minerals that make up the human body, 99% of which is used for the construction of bones and teeth, and the remaining 1% is the activity of various enzymes. It plays an important role in life activities such as expression, muscle storage, calming of cell excitement or blood coagulation.

For example, pancreatic juice produced in the human pancreas and assisting in digestion contains a high concentration of calcium. However, if the balance of the living body is lost for any reason, crystallization of calcium carbonate occurs. You can make stones. In addition to the pancreas, calcium calculus occurs in various organs in the body, which adversely affects the human body. Therefore, development of a substance that suppresses calcium crystallization that can be used for prevention and treatment of calculus has been awaited. I have.

[0004] Although it is such an important calcium,
Looking at its intake, it is said that the required amount for Japanese is 600 mg per adult per day, but according to the results of the 1994 National Nutrition Survey by the Ministry of Health and Health, the actual intake was The actual amount is 545 mg, which is lower than the required amount. Insufficiency of calcium is known to cause serious diseases such as osteoporosis and hypertension, and insufficiency of calcium has become a social problem. Furthermore, calcium ingested in the gastrointestinal tract as food is absorbed into the blood from the intestinal tract by a complicated mechanism, but the absorption rate of calcium salts and calcium agents in the intestinal tract is 5%.
There is also a report that it is 0% or less, and more than half is excreted outside the body without being absorbed. For this reason, substances that enhance calcium absorption in the intestinal tract have been developed.

[0005] As one of them, casein phosphopeptide (CPP) has been developed. CPP is a phosphopeptide obtained by hydrolyzing casein with trypsin and hydrolyzing it, and binds to calcium to form a soluble complex. For this reason, it is considered that calcium is solubilized by suppressing precipitation of calcium in an aqueous solution to increase the absorption rate of calcium (Japan Food Science, Vol. 1, pp. 21-32 [1990 ];
JP-A-58-170440; JP-A-7-2411
No. 72). However, since CPP is an enzymatically decomposed product of casein, it is necessary to carry out an enzymatic reaction of casein, which is a raw material, and the peptide which is a by-product of enzymatic decomposition has a bitter taste. It has several problems such as the necessity of sufficiently separating peptides, and is very expensive.

[0006] Poly-L-glutamic acid also has an effect of increasing calcium absorption in the intestinal tract (Biosci. Biotech. Bioche.
m., Vol. 58, pp. 1662-1665 [1994]), but it is a synthetic product and is not permitted as a food additive, and is used for safety and other purposes. It has not been. Further, poly-γ-glutamic acid produced by a microorganism (JP-A-3-30648) is inconvenient to handle because it has low calcium crystallization inhibitory activity and extremely high solution viscosity.

[0007] Further, as a substance which promotes absorption of calcium, a peptide derived from bone (JP-A-4-16165) and a substance containing butyric acid as a basic component (JP-A-4-108)
No. 360 gazette), however, these have problems in production and utilization, and have not been put to practical use.

[0008]

SUMMARY OF THE INVENTION The present invention solves the above-mentioned problems of the prior art, suppresses calcium crystallization in the body, and promotes calcium crystallization suppression / absorption that promotes absorption of calcium in the intestinal tract. An object of the present invention is to provide an accelerator, food and drink containing the same, and feed.

[0009]

Means for Solving the Problems The inventors of the present invention have conducted intensive studies to solve the above-mentioned problems, and as a result, they have been deposited with bacteria, especially at the Biotechnology Industrial Technology Research Institute inhabiting Lake Vanda in Antarctica. The present inventors have found that proteins obtained from the cells of four types of novel bacteria suppress calcium crystallization and thereby promote calcium absorption, and completed the present invention based on the findings. .

That is, the calcium crystallization inhibitor / absorption enhancer according to the present invention is deposited on a bacterium having a calcium crystallization inhibitory effect, in particular, deposited on the Biotechnology Industrial Research Institute.
A protein obtained from cells of novel kinds of bacteria is used as an active ingredient.

The food and drink and feed of the present invention contain the above-mentioned calcium crystallization inhibitor / absorption promoter.

[0012]

DETAILED DESCRIPTION OF THE INVENTION In detail, the present inventors have found that calcium is located near the Pacific Ocean shore of Antarctica, which is covered with ice, which is 30 meters thick year-round. Four Gram-positive bacteria having calcium crystallization inhibitory activity were isolated from Lake Vanda with a very high concentration. Those four
As a result of examining the taxonomic properties of the bacteria having the highest calcium crystallization inhibitory effect among the bacteria of the strain, they were identified as the genus Bacillus and designated as Bacillus species 20-1 strain. The bacterium having the calcium crystallization inhibitory activity of the present invention, including the bacterium, is Bacillus species 20-1 (accession number FERM).
P-17193), Strain19 (Accession number FERM P)
-17194), Strain 20 (Accession number FERMP)
-17195) and Strain 25W (Accession No. FERM)
P-17196) and deposited under the above accession number with the Institute of Biotechnology and Industrial Technology, Ministry of International Trade and Industry.

The bacteriological properties are shown in Table 1 below. Tests and classification of the bacteriological properties of these strains were carried out according to the following documents. Bergey's Manual of Systematic Bacteriology
Bacteriology], 2nd edition (1986) Manual for the Identification of Medical Bacteria [Manual for the Identificati]
on of Medical Bacteria], 2nd edition (1983)

The results show that Bacillus species 20-1
The strain was a spore-forming gram-positive bacillus, motility, growth under aerobic conditions, positive catalase activity, and acid production from glucose, and was identified as belonging to the genus Bacillus. In addition, oxidase activity negative, growth under anaerobic conditions, Voges-Proskauer test negative, generates acid from arabinose, does not generate acid from xylose or mannitol, does not grow at 5 ° C, but 10 ° C and 30 ° C
Growth at pH 6.8, with starch resolution,
Does not use citrate. The mycological properties as described above were exhibited.

Further, three other strains (Strain 19, Strat 19)
The following microbiological properties of S. in 20, Strain 25W) are shown. The bacteria are Gram-positive bacteria that form spores, which grow under aerobic conditions, catalase activity positive, grow under anaerobic conditions, and Voges- Proskauer test negative, produces acid from glucose, does not produce acid from arabinose, xylose, mannitol and does not utilize citrate. Strain 20 strain is positive for oxidase activity,
Strain 19 strain and Strain 25W strain were negative. However, from these properties, the genus of the above three strains,
The species could not be identified and is still unknown.

[0016]

[Table 1]

As a method for extracting a protein obtained from the cells of the bacterium of the present invention, a commonly used cell disruption method can be used. For example, a method using a pressurized cell disruption device such as a French press or X-press,
Mechanical grinding using a ball mill, Dyno mill, etc., sonication, homogenizer, freeze-thaw, osmotic treatment, enzyme treatment using lysozyme, etc. Can also be extracted. In consideration of protein stability and extraction efficiency, it is desirable to use a physical disruption method such as pressurized cell disruption, mechanical attrition, ultrasonic treatment, and a homogenizer. The protein extract is
Removal of nucleic acid by protamine sulfate or streptomycin sulfate, etc., salting out of ammonium sulfate or the like, precipitation with an organic solvent such as ethanol, fractionation by isoelectric precipitation, ion exchange, adsorption, gel filtration, hydrophobic or affinity as required Purification, or dialysis or concentration steps may be performed.

The protein extract of the present invention obtained by the above method was found to inhibit calcium crystallization at a protein concentration of 0.04 to 3.0 μg / ml. In addition, when the extract was treated with Proteinase K, a decrease in the activity was observed, so it was confirmed that the center of the activity was a protein.

As the calcium crystallization inhibitor / absorption promoter of the present invention, the extract prepared by the above method may be used as it is, but generally, it is dissolved or dispersed in a suitable liquid carrier, or Emulsifiers, dispersants, suspending agents, spreading agents, penetrating agents, wetting agents, stabilizers, etc. are further added to these, if desired, mixed with or adsorbed on a powder carrier. In the form of preparations such as preparations, capsules, tablets, powders, etc., used for prevention and treatment of pancreatic stones for the purpose of suppressing calcium crystallization, and for prevention and treatment of osteoporosis for the purpose of promoting calcium absorption be able to.

The foods and drinks containing the calcium crystallization inhibitor / absorption promoter of the present invention include various foods and drinks such as soft drinks, fruit juice drinks, fermented drinks, drinks such as milk, chewing gum, candy, and the like. Tablet confections, gummy jellies, biscuits, confectionery such as chocolate, ice cream, frozen desserts such as frozen desserts, dairy products such as yogurt and cheese, meat products such as ham and sausage, fish paste products such as kamaboko and chikuwa, bread, hot cakes, various types Side dishes,
Pudding, soup and the like.

Further, the feed of the present invention, such as pet food such as dock food and cat food, and feed for livestock, is a feed containing the above calcium crystallization inhibitor / absorption promoter.

When the calcium crystallization inhibitor / absorption promoter of the present invention is used particularly for the purpose of promoting calcium absorption, a calcium-containing raw material such as milk,
When it is blended with dairy products such as yogurt and cheese and does not contain calcium, it can be further blended with calcium to further promote the effect of promoting calcium absorption. In each case, the ratio was calculated as calcium crystallization inhibitory protein extract / calcium (weight /
(Weight) is preferably 0.005 or more.

The effective amount of the calcium crystallization inhibitor / absorption enhancer of the present invention is difficult to define unequivocally. To demonstrate the absorption promotion effect,
The amount is suitably 0.08 mg / kg / day or more, preferably 0.1 to 100 mg / kg / day.

Similarly, for the purpose of preventing and treating calculi in the body, it is also preferable to take 0.01 to 100 mg / kg / day.

Hereinafter, the present invention will be described in more detail with reference to Examples.

[0026]

【Example】

Example 1 Preparation of Calcium Crystallization Inhibiting Protein Bacillus species 20-1 was pre-cultured in trypticase soy broth (TSB) medium at 18 ° C. for 24 hours.
Main culture was performed at 25 ° C. for 48 hours in a liquid medium in which 0.5% (w / v) CaCl ₂ .2H ₂ O was added to the SB medium. The cells were collected, and 10 mM Tris-HCl buffer (pH 8.
7) twice, and about twice the amount of 0.5 M EDTA (p
H8.0) The suspension was suspended in an aqueous solution and crushed by a French press (pressure-type cell disruption apparatus) for 30 minutes in a cycle of 30 seconds. Thereafter, streptomycin sulfate 1.5 times the protein concentration of the cell extract was added, and ultracentrifugation (10%) was performed.
000 × g, 4 ° C., 2 hours) to remove nucleic acids. The supernatant is dialyzed (4 ° C.) against distilled water and PEG6000
And concentrated to 600 μg / ml or more as a protein, followed by ammonium sulfate salting out.
-1 strain extracted protein was obtained.

[0027]

Example 2 Preparation of Calcium Crystallization Inhibiting Protein The Bacillus species 20-1 strain extracted protein obtained in Example 1 was further purified. Ethanol cooled to −20 ° C. was added to the extracted protein of the 20-1 strain, which was concentrated to a protein concentration of 21.4 mg / ml or more using PEG6000 so as to be 60% of the total volume, and then left for 30 minutes, and then centrifuged. Separation (27,700 ×
g, 4 ° C, 30 minutes). Furthermore, in the supernatant -2
After adding ethanol cooled to 0 ° C. to 80% of the total volume, the mixture was allowed to stand for 30 minutes, and then centrifuged (27,7).
00 × g, 4 ° C., 30 minutes). Small amount of 10 mM
After suspending in a Tris-HCl buffer (pH 8.7), the suspension was dialyzed against distilled water (4 ° C.), and concentrated to a protein concentration of 1.99 mg / ml with PEG6000 to further purify Bacillus species 20-. One strain extracted protein was obtained.

[0028]

Example 3 Measurement of Calcium Crystallization Inhibiting Effect C was determined from the reaction between an aqueous NaHCO ₃ solution and an aqueous CaCl ₂ solution.
Reaction to precipitate aCO ₃ (NaHCO ₃ + CaCl ₂ →
CaCO ₃ + HCl + NaCl) was used to determine the effect of inhibiting calcium crystallization.

That is, 30 μl of the protein extract was added to an aqueous sodium hydrogen carbonate solution (1.5 ml) adjusted to 20 mM and pH 8.7, and the mixture was stirred well with a stirrer. Thereafter, an aqueous calcium chloride solution (1.5 ml) adjusted to 20 mM and pH 8.7 was added thereto, and the mixture was reacted at 25 ° C. During the course of the reaction, the absorbance at a wavelength of 570 nm was measured over time.

As the final protein concentration, Bacillus speci
es 20-1 strain extracted protein at 0.5 μg / ml,
Those containing 2.0 μg / ml and those to which distilled water was added as a control were tested for the effect of inhibiting calcium crystallization, and the results are shown in FIG.

In the case of distilled water, a sharp increase in absorbance was observed by about 200 seconds, and the maximum value was observed after about 250 seconds, indicating that the reaction was completed. When the protein extracted from the 20-1 strain (final protein concentration: 2.0 μg / ml) was added, no increase in absorbance was observed, and the crystallization of calcium was completely suppressed. When the protein extracted from the 20-1 strain (final protein concentration: 0.5 μg / ml) was added, the nucleation of calcium crystals was delayed.

[0032]

Example 4 Measurement of Calcium Crystallization Suppressing Effect According to the method for measuring calcium crystallization suppressing effect of Example 3,
With respect to the protein extracted from Bacillus species 20-1 strain, the calcium crystal inhibitory effect was tested by measuring the absorbance at a wavelength of 570 nm with time, and the results are shown in FIG. The final concentration of the protein extracted from the 20-1 strain was 0.04 μg / ml and 0.1 μg / ml.

In the case of the extracted protein of strain 20-1 (final protein concentration: 0.1 μg / ml), no increase in absorbance was observed, and crystallization of calcium was completely suppressed. Also,
20-1 strain extracted protein (final protein concentration 0.
04 μg / ml), a delay in the nucleation of calcium crystals was observed.

[0034]

Example 5 Measurement of Calcium Crystallization Inhibition Effect Next, according to the measurement method of Example 3, the reaction inhibition effect of a protein extracted from Bacillus species 20-1 strain during the progress of calcium crystallization reaction was tested. did. In the test, calcium crystallization was first advanced, and after 60 seconds, the protein extracted from the 20-1 strain was converted to a final protein concentration of 2.0 μg / m 2.
1 and the absorbance at a wavelength of 570 nm was measured over time. The result is shown in FIG.

When the protein extracted from the 20-1 strain was added, no subsequent increase in absorbance was observed, confirming that calcium crystallization was completely inhibited.

[0036]

Example 6 Comparison of calcium crystallization inhibitory effect with other substances From the results of Example 3, in the case of distilled water, a sharp increase in absorbance was observed after about 200 seconds, and the maximum value was observed after about 250 seconds. Indicated that the reaction was complete. Therefore, Bacillus species 2
0-1 strain extracted protein (2.0 μg / ml) and other substances, ie, casein phosphopeptide (CPPIII)
(2 μg / ml), poly-L-glutamic acid (2 μg / ml)
ml), poly-γ-glutamic acid (2 μg / ml), E
DTA (1.5 × 10 ⁻⁴ M), citric acid (0.4 × 1
0 ^-4 M), phosvitin (15μg / ml), heparin (15μg / ml), chondroitin sulfate C (15 [mu] g
/ Ml), albumin (10 μg / ml), lactoferrin (10 μg / ml), lipase (10 μg / m
l), trypsinogen (10 μg / ml), α-chymotrypsinogen A (10 μg / ml), α-amylase (10 μg / ml), esterase (10 μg / m
Regarding l), the inhibition rate was calculated from the absorbance 200 seconds after the reaction according to the following equation 1. The effect of inhibiting calcium crystallization with other substances was compared. Table 2 shows the results.

[0037]

(Equation 1)

[0038]

[Table 2]

As shown in Table 2, when protein extracted from Bacillus species 20-1 strain (final protein concentration: 2.0 μg / ml) prepared according to the method of Example 1, the crystallization inhibition rate was 100%. In comparison with other substances, 62.8% of casein phosphopeptide (CPPIII), which is known as a calcium solubilizer,
L-glutamic acid (manufactured by Sigma; No. P-4886, sodium salt) had an inhibition rate of 59.4%, and poly-γ-glutamic acid extracted from natto and precipitated by ethanol precipitation had a 49.0% inhibition rate. . Chelating agents such as EDTA and citric acid have 14.4% and 24.5% inhibition, respectively.
In addition, phosvitin, which is reported to inhibit crystallization of calcium carbonate, had an inhibition rate of 30.1%. In addition, no significant crystallization inhibitory effect was observed even when phosphoproteins and enzymes were added. Bacillus speci of the present invention
Cell proteins extracted from es 20-1 strain significantly inhibited calcium crystallization at low concentrations.

[0040]

Example 7 Comparison of Calcium Crystallization Inhibiting Effect with Other Bacteria According to the method of Example 6, in addition to Bacillus species 20-1 strain, Strain 19, Strain 20, and Strain 20 of the present invention were used.
25 W cell protein, as well as other bacteria, Baci
Bacillus subtilis of the genus llus, Strain 2, Strain 4-1, Strain 5, Str. isolated from Lake Vanda, Antarctica
Comparative tests were performed on ain 15 and Strain 17. The preparation of the protein was carried out according to Example 1. Table 3 shows the results.

[0041]

[Table 3]

As shown in Table 3, among the bacteria isolated from Lake Vanda in Antarctica, Bacillusspecies 20-1
In addition to strains, Strain 19, Strain 20 and Strain 2
It was confirmed that the 5 W bacterial cell protein also had a calcium crystallization inhibitory effect. However, from the same Antarctic
Strain 2, Strain 4-1, Strain 5, Strain 15
Strain 17 and Bacillus subtilis did not show any calcium crystallization inhibitory effect.

Further, the four kinds of bacteria (Bacillus s.
pecies 20-1 strain, Strain 19, Strain 20, St
rain 25W) is novel, and its calcium crystallization inhibitory effect is remarkably superior to the conventionally known ones. Therefore, proteins obtained from these cells are also expected to be novel proteins.

[0044]

Example 8 Chewing gum was prepared according to the following formulation. Gum base 20.0% Sugar 55.0% Glucose 23.7% Softener 1.0% Extracted protein 0.3%

[0045]

Example 9 A chewing gum was prepared according to the following formulation. Gum base 20.0% Xylitol 75.0% Reduced maltose 3.8% Softener 1.0% Extracted protein 0.2%

[0046]

Example 10 Tablets were prepared according to the following recipe. Sugar 75.0% Lactose 20.0% Glycerin fatty acid ester 0.2% Fragrance 0.4% Extracted protein 0.1% Purified water 4.3%

[0047]

Example 11 Chocolate was prepared according to the following recipe. Sugar 41.0% Cocoa mass 15.0% Whole milk powder 25.0% Cocoa butter 18.0% Emulsifier 0.3% Fragrance 0.4% Extracted protein 0.3%

[0048]

Example 12 A beverage was prepared according to the following formulation. Fructose dextrose liquid sugar 5.00% Sugar 4.50% Acidulant 1.28% Flavor 0.20% Extracted protein 0.02% Purified water 89.00%

[0049]

Example 13 A beverage was prepared according to the following formulation. Orange juice 85.25% Sugar 11.70% Citric acid 2.00% Flavor 1.00% Extracted protein 0.05%

[0050]

Example 14 An ice cream was prepared according to the following recipe. Fructose dextrose liquid sugar 0.5% Sugar 8.7% Acidulant 1.2% Fragrance 0.3% Purified water 89.0% Stabilizer 0.2% Extracted protein 0.1%

[0051]

Example 15 A dock food was prepared according to the following formulation. Corn 33.0% Flour 35.0% Soybean meal 21.0% Rice bran (defatted) 5.5% Meat meal 5.0% Mineral mix 0.2% Extracted protein 0.3%

[0052]

Example 16 A capsule was prepared according to the following formulation. Extracted protein 50.0% Lactose 48.0% Magnesium stearate 2.0%

The above components were uniformly mixed, and the mixed powder was filled in hard capsules.

[0054]

Example 17 An injection was prepared according to the following formulation. Extracted protein 0.05% Glucose 1.00% Water for injection 98.95%

After filtering the above mixed solution through a membrane filter, sterilization filtration was performed again. The filtrate was aseptically dispensed into vials, filled with nitrogen gas, and sealed to obtain an injection.

[0056]

Example 18 A tablet was prepared according to the following formulation. Extracted protein 20.0% Fine granules for direct hit No.209 (Fuji Chemical Co., Ltd.) 48.0% Crystalline cellulose 30.0% Magnesium stearate 2.0%

The above components were uniformly mixed, and the resulting mixture was tabletted to give a tablet of 200 mg per tablet.

Fine particles for direct hit No. 209 (magnesium aluminate metasilicate 20%, corn starch 30%, lactose 50%)

[0059]

Example 19 A syrup was prepared according to the following formulation. Extracted protein 0.1% Simple syrup 30.0% Purified water 69.9%

The extracted protein was completely dissolved in purified water, and a single syrup was added and mixed to obtain a syrup.

[0061]

Example 20 A candy was prepared according to the following formulation. Extracted protein 0.2% sugar 50.0% starch syrup 35.3% flavor 0.5% purified water 14.0%

[0062]

Example 21 A biscuit was prepared according to the following recipe. Extracted protein 0.5% Flour 50.6% Corn starch 5.1% Sugar 12.7% Margarine 6.5% Salt 0.3% Sodium carbonate 1.3% Ammonium carbonate 0.5% Soy lecithin 0.3% Total Egg 4.1% Spice 0.3% Purified water 17.8%

The above materials were mixed to form a dough, which was spread, molded, and roasted in an oven to produce a biscuit.

In the following, for the purpose of promoting calcium absorption, calcium crystallization inhibition and calcium crystallization of the present invention are carried out together with calcium.
Examples of foods and drinks and feeds containing the absorption enhancer will be described.

[0065]

Example 22 A chewing gum was prepared according to the following recipe. Gum base 20.0% Sugar 55.0% Glucose 23.0% Softener 1.0% Calcium carbonate 0.5% Extracted protein 0.5%

[0066]

Example 23 A chewing gum was prepared according to the following formulation. Gum base 20.0% Sugar 75.0% Reduced maltose 3.6% Softener 1.0% Dibasic calcium phosphate 0.2% Extracted protein 0.2%

[0067]

Example 24 Tablets were prepared according to the following formulation. Sugar 74.0% Lactose 20.0% Glycerin fatty acid ester 0.2% Fragrance 0.4% Calcium carbonate 0.8% Extracted protein 0.3% Purified water 4.5%

[0068]

Example 25 A chocolate was prepared according to the following recipe. Sugar 41.0% cacao mass 15.0% whole milk powder 25.0% cocoa butter 18.0% calcium carbonate 0.3% emulsifier 0.3% fragrance 0.2% extracted protein 0.2%

[0069]

Example 26 A beverage was prepared according to the following formulation. Fructose dextrose liquid sugar 5.00% Sugar 4.50% Acidulant 1.27% Flavor 0.20% Extracted protein 0.02% Calcium chloride 0.01% Purified water 89.00%

[0070]

Example 27 A beverage was prepared according to the following formulation. Orange juice 85.20% Sugar 11.70% Citric acid 2.00% Flavor 1.00% Calcium chloride 0.05% Extracted protein 0.05%

[0071]

Example 28 An ice cream was prepared according to the following recipe. Fructose dextrose liquid sugar 0.3% Sugar 8.7% Acidulant 1.0% Flavor 0.2% Purified water 89.0% Stabilizer 0.3% Calcium chloride 0.2% Extracted protein 0.3%

[0072]

Example 29 A feed for laying hens was prepared according to the following formulation. Maize 51.0% Mylo 15.3% Soybean meal 17.0% Fish meal 3.3% Rice bran 8.2% Salt 0.2% Animal fats and oils 3.0% Vitamin mix 0.2% Calcium lactate 1.0% Extracted protein 0.8%

[0073]

Example 30 A capsule was prepared according to the following formulation. Extracted protein 50.0% Lactose 47.0% Dibasic calcium phosphate 1.0% Magnesium stearate 2.0%

The above components were uniformly mixed, and the mixed powder was filled in hard capsules.

[0075]

Example 31 An injection was prepared according to the following formulation. Extracted protein 0.05% Glucose 1.00% Second calcium phosphate 1.00% Water for injection 97.00%

After filtering the above mixed solution through a membrane filter, sterilization filtration was performed again. The filtrate was aseptically dispensed into vials, filled with nitrogen gas, and sealed to obtain an injection.

[0077]

Example 32 A tablet was prepared according to the following formulation. Extracted protein 20.0% Fine granules for direct hit No.209 (Fuji Chemical Co., Ltd.) 37.0% Crystalline cellulose 33.0% CMC calcium 8.0% Magnesium stearate 2.0%

The above components were uniformly mixed, and the mixed powder was tableted to give a tablet of 200 mg per tablet.

No. 209 for direct punching (magnesium aluminate metasilicate 20%, corn starch 30%, lactose 50%)

[0080]

Example 33 A syrup was prepared according to the following formulation. Extracted protein 0.1% Simple syrup 30.0% Purified water 69.8% Calcium carbonate 0.1%

The extracted protein was completely dissolved in purified water, syrup was added and mixed to obtain a syrup.

[0082]

EFFECT OF THE INVENTION The protein obtained from the bacterial cells of the present invention has remarkably superior calcium crystallization inhibitory and absorption promoting effects as compared with the substances obtained by the prior art. The calcium crystallization inhibitor / absorption enhancer of the present invention, and foods, drinks and feeds containing the same exhibit excellent calcium crystallization inhibitory / absorption promoting effects.

[Brief description of the drawings]

FIG. 1. Bacillus species for calcium crystallization
It is the graph which examined the influence of 20-1 strain extraction protein concentration.

FIG. 2. Bacillus species for calcium crystallization
It is the graph which examined the influence of 20-1 strain extraction protein concentration.

FIG. 3. Bacillus species for calcium crystal growth
It is the graph which examined the inhibitory effect of 20-1 strain extraction protein.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61P 3/00 A61K 31/00 603 43/00 643D A61K 35/74 35/74 D C12N 1/20 C12N 1 / 20 A // (C12N 1/20 C12R 1:07) (72) Inventor Hidehisa Kawahara 7-11-34, Nagaomotomachi, Hirakata City, Osaka Prefecture No. 1 (72) Inventor Yoji Saeki 1-44 Haruno, Omiya-shi, Saitama Urban Mirai No. 806 No. 806 F-term (reference) 2B150 AA01 AA06 AB01 AB20 AC02 DC23 DD11 DD26 DD61 DH04 4B018 LB01 LB08 MD04 MD20 MD85 ME05 MF01 4B065 AA15X AC20 BA22 BB02 BC02 BC03 BD01 BD15 CA41 CA43 4C084 AA02 BA03 BA44 CA04 DC50 MA02 MA17 MA23 MA35 MA37 MA52 MA66 ZC212 4C087 AA01 AA02 BC64 CA16 MA02 MA52 ZC21

Claims (6)

[Claims] 1. A calcium crystallization inhibitor / absorption promoter comprising, as an active ingredient, a protein obtained from a bacterial cell having a calcium crystallization inhibitory action. 2. The calcium crystallization inhibitor / absorption promoter according to claim 1, wherein the bacterium is one of the following four types of bacteria deposited at the Institute of Biotechnology and Industrial Technology. Bacillus species 20-1 (Accession number FERM P-
17193) Strain 19 (Accession No. FERM P-17194) Strain 20 (Accession No. FERM P-17195) and Strain 25W (Accession No. FERM P-17196) 3. A food or drink comprising the calcium crystallization inhibitor / absorption enhancer according to claim 1 or 2. 4. A feed comprising the calcium crystallization inhibitor / absorption promoter according to claim 1 or 2. 5. The food or drink according to claim 3, further comprising calcium. 6. The feed according to claim 4, further comprising calcium.