JP2002053472A - Lipid peroxidation inhibitor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a lipid peroxidation inhibitor that is useful as a prophylactic or therapeutic for intestinal mucus disorder, for example, ulcerative colitis, large intestine cancer and the like and for aging, because the inhibitor can reduce the oxidative stress to intestinal mucous membrane and can become commercial items suitable for increasing the body healthiness by preventing and improving the intestinal diseases and ageing in the present time that the damage to intestinal mucous membrane can be inhibited without inhibition of internal absorption of iron ions with high safety and the iron supplement food and iron ion preparations are daily taken. SOLUTION: This lipid peroxidation inhibitor comprises the cell bodies or the constitution components of the cell bodies of a microorganism belonging to Bifidobacterium or Streptococcus as an active ingredient. This invention additionally relates to prophylactics and therapeutics for damages of intestinal mucous membrane including the peroxidation inhibitor and health foods and beverages containing the same.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a lipid peroxidation inhibitor useful as a food or drink or a pharmaceutical for the purpose of preventing or improving intestinal mucosal injury.

[0002]

2. Description of the Related Art In vivo lipids include hydroxyl radicals,
It is peroxidized by active oxygen such as peroxy radical and hydroperoxy radical and causes injuries such as inflammation, cancer and aging. It is known that a bivalent iron ion acts as a catalyst in such an active oxygen generation reaction as shown in the following formulas (1) to (5).

[0003]

Embedded image

[0004] [in the formula, HO ^· is hydroxy radicals, LOOH
Lipid peroxidation, LO is alkoxy anion, LOO ^· the peroxy radicals, HOO ^· the hydroperoxy radicals, O ₂ ^{- ·}
Represents a superoxide anion radical, respectively. ]

[0005] In contrast, there are iron ion oxidases such as ferroxidase and iron-binding proteins such as transferrin and ferritin in the living body, and a mechanism for stabilizing iron ions and suppressing the reactivity thereof. Exists. However,
In the intestinal lumen, there is no such mechanism for inactivating iron ions, and there is an environment where highly reactive divalent iron ions are easily generated and presents a unique environment that is constantly exposed to oxidative load. I have. In other words, in the intestinal tract, oxygen supplied to the intestinal mucosa by blood flow is reduced by divalent iron ions to form a superoxide anion radical, and a superoxide anion radical is also produced from intestinal bacteria, and this superoxide anion is produced. It is believed that radicals are converted to hydrogen peroxide by a disproportionation reaction, which further reacts with iron ions on the surface of the intestinal mucosa to generate hydroxyl radicals, which leads to peroxidation of lipids (see FIG. 1, CFBab
bs, 1990, Free Rad. Biol. Med., 12, 161-168), which largely involves divalent iron ions.

[0006] Therefore, in order to prevent peroxidation of lipids in the intestinal mucosal tissue, it is necessary to control the mechanism of free radical generation involving iron ions. For example, wheat bran containing phytic acid which chelate iron ions is required. It is also conceivable to actively administer rice bran and the like. However, iron is one of the minerals that is often deficient in infants, adolescents, men and women of childbearing age, and iron deficiency is a problem in pregnant women when giving birth to low-weight or premature babies and giving birth. Infants and children suffer from mental activity and cognitive impairment, and adults suffer from reduced work abilities. Therefore, it is indispensable for people with iron deficiency to actively supplement iron with various foods, drinks and medicines containing iron ions, and ingest substances such as phytic acid that inhibit the absorption of iron ions. It is not preferable.

On the other hand, vitamin E and its derivatives, which are antioxidants, are used as substances to prevent the formation of lipid peroxides. However, there were problems in processing characteristics and stability. Furthermore, it has been reported that Lactobacillus bacteria have an effect of suppressing oxidative damage to liver and erythrocytes (Japanese Patent Application Laid-Open No. 4-264034), but the effect of suppressing lipid peroxidation in the digestive tract is considered to be sufficient. I can't say.

[0008]

DISCLOSURE OF THE INVENTION The present invention can suppress the generation of lipid peroxide without affecting the absorption of iron ions in the intestinal tract, and can prevent or improve the effect of intestinal mucosal damage associated with lipid peroxidation. It is intended to provide foods and beverages or pharmaceuticals having the same.

[0009]

Means for Solving the Problems The present inventors have conducted various studies on substances that suppress the production of lipid peroxide, and found that cells of specific microorganisms or their constituents do not affect free iron ions. The present inventors have found that the production of lipid peroxide can be suppressed and that it can be used as a food or drink or a pharmaceutical product, and thus completed the present invention.

[0010] That is, the present invention provides a lipid peroxidation inhibitor comprising, as an active ingredient, cells of a microorganism belonging to the genus Bifidobacterium or Streptococcus.

[0011] The present invention also provides a preventive and therapeutic agent for intestinal mucosal injury containing the lipid peroxidation inhibitor.

Further, the present invention provides a food or drink containing the lipid peroxidation inhibitor.

[0013]

BEST MODE FOR CARRYING OUT THE INVENTION The lipid peroxidation inhibitor of the present invention is
It refers to a substance that controls peroxidation of lipids in a living body caused by free radicals such as active oxygen, in particular, peroxidation induced by iron ions, and suppresses the production of lipid peroxides.

Examples of the microorganism belonging to the genus Bifidobacterium used in the present invention include, for example, Bifidobacterium breve and Bifidobacterium longam.
gum), Bifidobacterium adressentis (Bif)
adolescentis), Bifidobacterium infantis, Bifidobacterium bifidum
and Bifidobacterium bifidum, and more preferably Bifidobacterium bifidum, and especially Bifidobacterium bifidum YIT deposited as Biotechnology Industrial Technology Research Institute Article 791 (FERM BP-791).
4007 is preferred.

The microorganism belonging to the genus Streptococcus includes, for example, Streptococcus thermophilus, Streptococcus salivarius, Streptococcus mitis and Streptococcus mitis. It is more preferable, and in particular, Bacteria No. 11891 (FERM P-11891) and 1
Streptococcus thermophilus YIT 2 deposited as No. 8189 (FERM P-18189)
001 is preferred. All of such microorganisms have been eaten since ancient times as fermented milk or viable bacterial preparations, and are safe to ingest at all times.

The microbial cells in the present invention may be, for example, lyophilized microbial cells, heat-treated dead cells, or their crushed cells, in addition to using the above-mentioned microbial cells as they are or using crushed cells. A processed product of a fungus body such as a product can also be used. Such microbial cells can be obtained by culturing a microorganism belonging to the genus Bifidobacterium or Streptococcus in a complex medium containing polyppetone or yeast extract or in milk.

[0017] As the cell component, for example, a fraction obtained by extracting cells or crushed cells with water or another solvent, or a fraction obtained by enzymatic treatment with a cell wall lysing enzyme, a nuclease, or a protease is used. Specifically, a water-soluble fraction obtained by hot-water extraction, a protoplast fraction from which the cell wall portion has been removed by cell wall lysing enzyme treatment, a cell wall lysing enzyme-soluble fraction of bacterial cells, and further a bacterial cell or A cell membrane fraction obtained by treating the protoplast with an organic solvent can be exemplified. These cell components are
It can be obtained by a generally used method.

Among the cells or cell components, the cells or cell lysate, the fraction containing cell membrane components, the protoplast fraction, or the cell membrane fraction obtained by treating the cells or protoplasts with an organic solvent are subjected to lipid peroxidation. It is particularly preferred because of its high inhibitory effect.

The cells thus obtained or the cell components thereof significantly suppress the lipid peroxidation induced by iron ions without affecting iron ions, as shown in the Examples below. It can be used safely for pregnant women and children who are concerned about iron deficiency. In addition, since the microorganisms are safe even when ingested at all times, they can be used as medicines for the purpose of preventing or treating intestinal mucosal damage and aging due to lipid peroxidation, and can be ingested daily as food and drink. And prevent intestinal mucosal disease and aging while supplementing iron ions. In view of the suppression of iron absorption, the cells used or the cell components used preferably have a reduction rate of the concentration of free iron ions in the intestinal tract of less than 10%, particularly less than 5%.

The lipid peroxidation inhibitor of the present invention can be made into various types of pharmaceutical compositions together with a pharmaceutically acceptable carrier according to a conventional method. For example, in the above cells or cell components, lactose, sucrose, sodium chloride, glucose, urea,
Excipients such as starch, calcium carbonate, kaolin, crystalline cellulose, silicic acid, etc., water, ethanol, propanol, glucose solution, starch solution, gelatin solution, binders such as carboxymethylcellulose, methylcellulose, potassium phosphate, polyvinylpyrrolidone, alginic acid Disintegrants such as sodium, agar powder, sodium hydrogen carbonate, calcium carbonate, polyoxyethylene sorbitan fatty acid esters, sodium lauryl sulfate, glycerin,
A humectant such as starch, a lubricant such as purified talc, stearate, polyethylene glycol and the like are added, and granules, tablets, capsules and the like can be produced by a conventional method.
Further, the tablet can be made into a tablet coated with a usual coating, if necessary, such as a sugar-coated tablet, a gelatin-encapsulated tablet, an enteric-coated tablet, a film-coated tablet or a double tablet or a multilayer tablet.

[0021] Further, the cells can be used as foods and drinks by directly adding the cells or cell components to the food, or by producing fermented foods such as fermented milk using the strain. Preferred examples of foods and drinks include fermented milk, dairy lactic acid bacteria drinks, fruit juice drinks, soups, crackers, cookies and the like. In particular, it is preferable to add microbial cells or microbial components to the iron-enriched food or beverage in which iron is enriched in the above-mentioned food or beverage because peroxidation of lipids or the like can be suppressed without inhibiting iron absorption. In addition, fermented milk which is easy to drink continuously and has a high lipid peroxide suppressing effect is particularly preferable as a form of food or drink. The food and drink also include animal feed.

[0022] Such foods and drinks may further contain various materials commonly used as foods. Specifically, sugars such as glucose, sucrose, fructose and honey, sugar alcohols such as sorbitol, xylitol, erythritol, lactitol and palatinit, emulsifiers such as sucrose fatty acid esters, polyglycerin fatty acid esters, lecithin, pectin, carboxymethyl cellulose And water-soluble soybean polysaccharides, stabilizers such as gellan gum, gum arabic, xanthan gum, carrageenan, guar gum and the like. In addition, various vitamins such as vitamin A, vitamin B, vitamin C and vitamin E, minerals such as calcium lactate, calcium gluconate, calcium pantothenate, various magnesium and zinc compounds, herbal extracts and the like are blended. It is also possible.

The amount of microbial cells or microbial constituents to be incorporated in the above-mentioned various preparations or foods and drinks cannot be determined unconditionally because they vary depending on the strain to be used or the condition, age, body weight, etc. of the patient to be taken. However, it is usually about 10 ⁹ to 10 ¹⁴ colony forming units (CFU) as viable bacteria per adult day, and about 0.01 to 30 g, preferably about 0.1 to 5 g, as dry cells.

[0024] In addition, when blended with a food or drink or a pharmaceutical composition enriched with iron, it is desirable to determine the amount of the cells or the components of the cells to satisfy the above-mentioned daily intake. In this case, the amount of iron is 0.1 to 10 mg of iron per day in the case of food or drink for the purpose of preventing iron deficiency,
In the case of a pharmaceutical composition for the treatment of anemia or the like, it is usually preferably 5 to 200 mg depending on the patient's condition.

[0025]

The present invention will be described in more detail with reference to the following examples.

Example 1 Production of Microbial Cells 1% of a cryopreserved strain was inoculated into a modified GAM bouillon medium (manufactured by Nissui Pharmaceutical Co., Ltd.) supplemented with 1% of lactose.
The culture was allowed to stand for 4 hours. 10 mL of this culture was inoculated into 1 L of a modified GAM broth medium supplemented with 2% lactose, and then incubated at 37 ° C.
The culture was allowed to stand for 4 hours. The microbial cells of the genus Bifidobacterium were anaerobically cultured in the presence of Anelopack Kenki (oxygen absorbing / carbon dioxide generating agent, manufactured by Mitsubishi Gas Chemical Co., Ltd.). After completion of the culture, the cells were collected by centrifugation at 4000 rpm for 10 minutes, and washed twice with physiological saline to obtain microbial cells (live cells). The cell yield after lyophilization was 1.3 for Bifidobacterium bifidum YIT 4007.
g, Streptococcus thermophilus YIT 20
01 was 1.0 g.

Example 2 Effect of Biological Membrane on Oxidative Injury As a system for evaluating lipid peroxidation reaction in the intestinal mucosa, a phospholipid liposome which is a model of a biological membrane was used as a reaction substrate, and divalent iron ions were added thereto. Then, a reaction system for initiating a peroxidation reaction was employed. This reaction system reproduces a reaction scheme in which hydrogen peroxide and divalent iron ions approach epithelial cells of the intestinal tract from the luminal side and cause oxidative damage to biological membranes (FIG. 1). The production of hydrogen peroxide depends on the reduction of oxygen molecules by divalent iron and the disproportionation reaction of supersquisid radicals as in the case of intestinal mucosa. The preventive and therapeutic effects of various bacterial cells on intestinal mucosal injury were evaluated by measuring the inhibition rate of oxidative damage (production of lipid peroxide) of biological membranes in this reaction system.

(1) Test method Preparation method of liposome solution 100 mg of egg yolk-derived L-α-phosphatidylcholine (Type XV-E; manufactured by Sigma) was dissolved in 10 mL of diethyl ether, and 0.6 mL of distilled water was added. The diethyl ether was evaporated in the evaporator during the treatment. This was combined with 0.1 M N- (2-acetamido) iminodiacetic acid (A
DA) 30 mL of a buffer solution (pH 6.7) was added, followed by sonication for 15 minutes to obtain a suspension. This suspension was added to 1500
The residue was removed by centrifugation at 5 rpm for 5 minutes to prepare about 25 mL of a liposome solution.

Method for Lipid Peroxide Production Reaction The microbial cells (live cells) shown in Table 1 were suspended in 0.1 M ADA buffer (pH 6.7) four times the wet weight, and further suspended in the same buffer. Serial dilutions were performed to prepare bacterial solutions having different bacterial cell concentrations (final concentration in the reaction solution: 10 ⁷ -10 ¹⁰ CFU / mL). This bacterial solution was added to 1.5 mL of the liposome solution to prepare a total of 1.98 mL of a reaction solution. 0.1 mM
0.1 mL of an aqueous solution of ferrous chloride and 0.02 mL of an aqueous solution of 0.1 M sodium ascorbate were added and reacted at 37 ° C. for 2 hours. The reaction was terminated by cooling the reaction vessel with ice water, and the lipid peroxide concentration was measured immediately.

Method for Measuring the Production of Lipid Peroxide Lipid peroxide was quantified as TBARS (thiobarbituric acid-reactive substance). The measurement procedure is described below. Acetic acid 80
A solution prepared by mixing mL and 320 mL of distilled water and adjusting the pH to 3.5 with 10 N sodium hydroxide was used as a buffer for TBARS measurement. Aliquot 0.1 mL of the reaction solution into a screw-mouth test tube, 0.2 mL of 8.1% sodium dodecyl sulfate, TBA
0.5 mL of buffer solution for RS measurement, 0.05 mL of 0.8% butylhydroxytolueneacetic acid solution, 1.0 mL of 0.8% thiobarbituric acid solution, 0.7 mL of 5 mM ethylenediaminetetraacetic acid solution, and 1.5 mL of distilled water They were mixed sequentially. The mixture was allowed to stand at 4 ° C. for 1 hour, heated in a boiling water bath for 1 hour, and cooled in ice water. 1.0 mL of distilled water and 5.0 mL of a mixed solution of n-butanol and pyridine (15: 1) were added thereto,
TBARS was extracted into the n-butanol / pyricin layer, and the fluorescence intensity (Ex. 515 nm, Em. 553 nm) of the extract was measured.

How to Obtain the Inhibition Rate of Lipid Peroxide Production Reaction The inhibition rate of the lipid peroxide production reaction was expressed as a percentage of the degree to which the production of lipid peroxide was inhibited by the addition of microbial cells. The calculation formula is shown below.

Lipid peroxide production inhibition rate (%) = (CT)
/ (CB) × 100 [where C is the amount of lipid peroxide when added without adding microbial cells (ferrous chloride added), and T is the peroxidation when added and added with microbial cells] The amount of lipid (with ferrous chloride) and B indicate the amount of lipid peroxide without ferrous chloride. ]

The inhibition rate of lipid peroxide production was measured by changing the bacterial cell concentration in the reaction solution, and the bacterial cell concentration when the inhibitory ratio reached 50% was determined and expressed as IC ₅₀ (CFU / mL). Exert effects at lower concentrations as those IC ₅₀ of less, indicating that the activity is stronger. The cell concentration was calculated from the number of colonies formed by culturing a modified GAM agar medium (manufactured by Nissui Pharmaceutical Co., Ltd.) coated with the bacterial solution at 37 ° C. for 2 days.

(2) Test Results As shown in Table 1, Bifidobacterium bifidum YIT 4007 and Streptococcus thermophilus YIT 2001 each had 2.0 × 10 ⁸ CF.
At the cell concentration of U / mL and 1.2 × 10 ⁸ CFU / mL, the production of lipid peroxide was inhibited by 50%. In contrast, the same lactic acid bacterium, Lactococcus lactis ATCC 1
9435, Lactobacillus buchineri YIT 007
7. Lactobacillus acidophil YIT 0070
Requires a cell concentration of 10 ⁹ CFU / mL or more, which is about 10 times, to exhibit the same activity.

[0035]

[Table 1]

From the above results, the action of various microorganisms to suppress the production of lipid peroxide induced by iron ions greatly differs between strains, and the activity of the microorganism of the present invention was far superior to that of other microorganisms. . In addition, Streptococcus, particularly Streptococcus thermophilus, was superior to Bifidobacterium.

Example 3 Effect of Biofilm on Oxidative Damage (Heat Treated Cells) The microbial cells prepared in Example 1 were autoclaved at 105 ° C. for 15 minutes to prepare heat treated cells. The lipid peroxide production inhibitory activity of the heat-treated cells was measured in the same manner as in Example 2, and compared with the activity of the live cells before the heat treatment. Table 2 shows the results.

[0038]

[Table 2]

As shown in Table 2, the heat-treated bacterial cells of Bifidobacterium bifidum YIT 4007 and Streptococcus thermophilus YIT 2001 retained the lipid peroxide production inhibitory activity of 80% or more of the viable cells. Therefore, when producing a pharmaceutical composition or a food or drink using these microorganisms, it is not always necessary to keep the cells alive, and the food or drink can be produced using a wider range of processing means.

Example 4 Effect on free iron ion concentration The production of lipid peroxide caused by the addition of iron
A reaction solution containing 0.1% inhibitory concentration of microbial cells or corn-derived sodium phytate (manufactured by Sigma) (0.1 M
An ADA buffer (pH 6.8), 35 mM sodium metabisulfite, and 48 μM ferrous chloride were reacted at 37 ° C. for 1 hour, and then centrifuged at 12,000 rpm for 10 minutes to collect a supernatant. To 1 mL of this supernatant was added 22 mM 2-nitroso-5- (N-
Propyl-N-sulfopropylamino) phenol
After 5 μL was added and the mixture was allowed to stand at room temperature for 15 minutes, the free iron ion in the solution was measured by measuring the absorbance at 750 nm. Table 3 shows the results.

[0041]

[Table 3]

As shown in Table 3, Bifidobacterium bifidum YIT 4007 and Streptococcus thermophilus YIT 2001 inhibited bacterial concentration by 50% (2.0 × 10 ⁸ C each).
FU / mL and 1.2 × 10 ⁸ CFU / mL) had no effect on the free iron ion concentration. On the other hand, sodium phytate reduced the free iron ion concentration by about 30% by the action of iron chelation at a concentration showing the same activity as these microbial cells. From the above results, it was found that Bifidobacterium bifidum YIT 4007 and Streptococcus thermophilus YIT 2001 do not have the effect of taking up (or adsorbing) iron ions into the microbial cells or the effect of iron chelation. Therefore, unlike phytic acid, the microorganism of the present invention can suppress only intestinal mucosal injury due to production of active oxygen without inhibiting absorption of iron ions.

Example 5 Effects on colonic mucosal injury (1) Test method Test feed Test feed having the composition shown in Table 4 (feed A: AIN-76 (recommended composition of the National Institute of Nutrition), feed B: feed A Feed containing 0.2% iron fumarate (iron content 0.07
%)), And feed B was administered to a 7-week-old BALB / c male mouse (manufactured by CLEA Japan) for 2 weeks, whereby FIG.
As shown in FIG. 7, the content of lipid peroxide in the colonic mucosa increased about 3-fold.

[0044]

[Table 4]

Animals and Breeding Methods Six-week-old BALB / c male mice (CLEA Japan) were acclimated and reared for one week, and then divided into groups of eight mice. To the test group, living cells (10 ⁹ CFU / day / animal) of Bifidobacterium bifidum YIT 4007 suspended in physiological saline were administered daily for 3 weeks using a gastric tube. The control group received physiological saline. Feed A was given for the first week and feed B for the next two weeks. Feed and water were freely available throughout the test period.

Measurement of Lipid Peroxide Content of Colon Mucosa Three weeks after the start of the test, the mice were dissected under pentobarbital anesthesia and the colon mucosa was collected. After washing the colonic mucosa with physiological saline, a 1.15% KCl solution 10 times the wet weight was added,
Homogenized with a Teflon (registered trademark) homogenizer while cooling with ice water. 0.1 mL of the colon mucosa homogenate was collected in a screw-mouth test tube, and TBARS was measured in the same manner as in Example 2. The TBARS value was expressed as malondialdehyde (MDA) equivalent per tissue protein mass. The protein content was measured using a BCA protein assay kit (Pierce).

(2) Test Results As shown in Table 5, the lipid peroxide content of the colonic mucosa of the group administered Bifidobacterium bifidum YIT 4007 was about 20% lower than that of the control group. During the breeding period, there was no significant difference in body weight between the two groups.

[0048]

[Table 5]

From the above results, it was confirmed that Bifidobacterium
Bifidum YIT 4007 had an effect of reducing oxidative damage of intestinal mucosa caused by iron ions.

Example 6 Effect on colonic mucosal injury (administration of mixed feed (1)) (1) Test method Test feed and group setting The test group consisted of a control group in which a feed of AIN-76 composition was loaded with 0.07% of iron, Bifidobacterium in the control diet
0.4% of freeze-dried cells of Bifidum YIT 4007
Or a 2.0% -added Bifidobacterium bifidum YIT 4007 administration group (B0.4% group and B2.0
% Group), a normal diet group without iron loading (iron content 0.0035)
%, And no cells).
The feed composition is shown in Table 6.

[0051]

[Table 6]

Animals and breeding method Six-week-old BALB / c male mice (manufactured by CLEA Japan) were acclimated and bred (MF feed freely) for 1 week, and then administered a test feed. At the start of the administration of the test feed, each individual was assigned to each group so that there was no weight difference between the groups. The administration period of the test feed was 2 weeks, and the feed and drinking water were freely taken. The breeding environment is a 12-hour light / dark cycle, a room temperature of 25 ° C. and a humidity of 55%.

Measurement of Lipid Peroxide Content of Colon Mucosa Two weeks after the start of the test, the mucosa was dissected under pentobarbital anesthesia, and the colon mucosa was collected. After washing the colonic mucosa with physiological saline, a 1.15% KCl solution 10 times the wet weight was added,
The mixture was homogenized with a Teflon homogenizer while cooling on ice. 0.1 mL of the colon mucosa homogenate was collected in a screw-mouth test tube, and TBARS was measured in the same manner as in Example 2. T
BARS values were expressed as malondialdehyde (MDA) equivalents per tissue protein mass. The protein content is B
The measurement was performed using a CA protein assay kit (Pierce).

Statistical analysis One-way analysis of variance was performed, and when a significant difference was observed, Tukey's multiple comparison was performed. The significance level was 5%.

(2) Test Results FIG. 3 shows the effect of the administration of Bifidecterium / Bifidum YIT 4007 in the diet on the oxidative damage of the colonic mucosa caused by iron ions. Addition amount of Bifidobacterium bifidum YIT 4007 cells 0.4
% And 2.0%, the lipid peroxide content of the colonic mucosa was significantly lower than that of the control group. During the breeding period, there was no significant difference in body weight between the four groups. From the above results, Bifidobacterium bifidum YIT 4007 had an effect of reducing oxidative damage to intestinal mucosa caused by iron ions.

Example 7 Effect on colonic mucosal injury (administration of mixed feed (2)) (1) Test method Test feed and group setting The test group was a control group in which 0.07% iron was loaded on a feed having an AIN-76 composition. 0.1% freeze-dried cells of Streptococcus thermophilus YIT 2001 were added to the group feed.
%, 0.4% or 2.0% added Streptococcus thermophilus YIT 2001 administration group (S 0.1
% Group, S 0.4% group, S 2.0% group) and a normal diet group not loaded with iron (iron content: 0.0035%, containing no bacteria), and each group had 12 animals. . In addition, each feed other than the normal diet group contains 2% of the feed weight of skim milk powder used as a protective agent for freeze-drying bacterial cells. Table 7 shows the feed composition.

[0057]

[Table 7]

Animals and Breeding Methods Six-week-old BALB / c male mice (manufactured by CLEA Japan) were acclimated and bred (MF feed freely) for one week, and then administered a test feed. At the start of the administration of the test feed, each individual was assigned to each group so that there was no weight difference between the groups. The administration period of the test feed was 2 weeks, and the feed and drinking water were freely taken. The breeding environment is a 12-hour light / dark cycle, a room temperature of 25 ° C. and a humidity of 55%.

Measurement of Lipid Peroxide Content of Colon Mucosa Two weeks after the start of the test, the mucosa was dissected under pentobarbital anesthesia and the colon mucosa was collected. After washing the colonic mucosa with physiological saline, a 1.15% KCl solution 10 times the wet weight was added,
The mixture was homogenized with a Teflon homogenizer while cooling on ice. 0.1 mL of the colon mucosa homogenate was taken into a screw-mouth test tube, and TBARS was measured in the same manner as in Example 2.
The TBARS value was expressed as malondialdehyde (MDA) equivalent per tissue protein mass. The protein content was measured using a BCA protein assay kit (Pierce).

Statistical Analysis A one-way analysis of variance was performed, and if a significant difference was found, Tukey's multiple comparison was performed. The significance level was 5%. (2) Test results Streptococcus thermophilus YIT 2001 against oxidative damage of colonic mucosa caused by iron ions
FIG. 4 shows the effect of the mixed feed administration. In both the 0.4% and 2.0% Streptococcus thermophilus YIT 2001 cell addition groups, the lipid peroxide content of the colonic mucosa was significantly reduced as compared to the control group, and the degree of the decrease was It was dose dependent. During the breeding period, there was no significant difference in body weight between the groups. Based on the above results, Streptococcus thermophilus YIT
2001 had the effect of reducing the oxidative damage of the intestinal mucosa caused by iron ions.

Example 8 Production of Food and Beverage Various edible compositions were produced using the microorganism of the present invention.
An example of the formulation is shown below.

(1) Fermented milk 10% skim milk powder is sterilized, and the microorganism of the present invention (Bifidobacterium bifidum YIT 4007 or Streptococcus thermophilus YIT 2001) is added to 2 parts of the present invention.
% Inoculated and cultured at 35 ° C. for 24 hours to produce fermented milk.

(2) Juice beverage The microorganism of the present invention (Bifidobacterium bifidum Y)
Using IT 4007 or Streptococcus thermophilus YIT 2001), a fruit juice beverage was produced according to the composition shown in Table 8.

[0064]

[Table 8]

(3) Beverages for Health The microorganism of the present invention (Bifidobacterium bifidum Y)
Using IT 4007 or Streptococcus thermophilus YIT 2001), a beverage for health was manufactured according to the composition shown in Table 9.

[0066]

[Table 9]

(4) Food for Health (Tablets) The microorganism of the present invention (Bifidobacterium bifidum Y)
Using IT 4007 or Streptococcus thermophilus YIT 2001), the compositions containing the additives shown in Table 10 were tableted to give tablets.

[0068]

[Table 10]

(5) Production of fermented milk drink (fermented milk) 20 parts by weight of skim milk powder was dissolved in 80 parts by weight of water and sterilized at 120 ° C. for 3 seconds, and then 2% of Streptococcus thermofils YIT 2001 strain, 2% 1.0% of Bacterium bifidum YIT 4007 was inoculated and cultured for 24 hours. This was homogenized with a homogenizer at 15 MPa to obtain fermented milk.

(Syrup solution)
It was dissolved in warm water at 0 ° C and sterilized at 120 ° C for 3 seconds to prepare a syrup solution.

[0071]

[Table 11]

After mixing 40 parts by weight of the fermented milk and 60 parts by weight of the syrup liquid, the mixture was filled in a polystyrene container and sealed to obtain a fermented milk product. The organoleptic evaluation of this fermented milk product showed that it had a good flavor and maintained high stability even after standing at 10 ° C. for one week.

(6) Production of Tablet According to the formulation shown in Table 12, various components were mixed and tableted to produce a tablet.

[0074]

[Table 12]

The tablets obtained had a good taste.

[0076]

Industrial Applicability The lipid peroxidation inhibitor of the present invention suppresses lipid peroxidation in vivo, particularly lipid peroxidation induced by iron ions, and reduces oxidative load on the intestinal mucosa and the like. It is useful as a preventive or therapeutic agent for intestinal mucosal damage such as ulcerative colitis and colon cancer and aging. In addition, since it can suppress intestinal mucosal injury without inhibiting absorption of iron ions into the body and is highly safe, it can be used safely for pregnant women and children, and iron supplements and iron preparations are commonly used Nowadays, it can be a product form suitable for the times as a means of preventing and improving intestinal diseases and aging and promoting health.

[Brief description of the drawings]

BRIEF DESCRIPTION OF DRAWINGS FIG. 1 is a diagram showing a production route of hydroxy radical in intestinal mucosa. In the figure, HO · is hydroxy radicals, O ₂ ^- · denotes a superoxide anion radical.

FIG. 2 shows feed A or feed B (iron fumarate added)
FIG. 2 is a graph showing the lipid peroxide content of the colonic mucosa after administration of for 2 weeks.

FIG. 3 shows Bifidobacterium bifidum Y
It is a figure which showed the lipid peroxide content of the colon mucosa at the time of IT 4007 feeding administration.

FIG. 4 is a graph showing the lipid peroxide content of the colonic mucosa when Streptococcus thermophilus YIT 2001 was administered as a feed.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61P 1/04 A61P 35/00 35/00 39/06 39/06 A23C 9/127 // A23C 9/127 A23L 2/00 F (72) Inventor Kenji Oishi 1-1-19 Higashi-Shimbashi, Minato-ku, Tokyo Yakult Honsha Co., Ltd. (72) Inventor Yasuhito Yoshida 1-1-19, Higashi-Shimbashi, Minato-ku, Tokyo Yakult Honsha (72) Inventor Kakee Yokoi 1-1-19 Higashi-Shimbashi, Minato-ku, Tokyo, Japan (72) Inventor Yasue Wada 1-1-19, Higashi-Shimbashi, Minato-ku, Tokyo, Japan No. Yakult Honsha Co., Ltd. (72) Inventor Joichi Watanabe 1-1-19 Higashi-Shimbashi, Minato-ku, Tokyo F-term (reference) 4B001 AC31 BC14 EC05 4B017 LC03 LG02 LG04 LK21 LL09 LP05 4B018 LE01 MD86 MD87 ME06 MF08 MF13 4C087 AA01 AA02 BC59 BC60 BC61 CA09 MA23 MA35 MA36 MA52 NA05 NA14 ZA66 ZA68 ZB26 ZC33 ZC37 ZC41

Claims (7)

[Claims] 1. A lipid peroxidation inhibitor comprising, as an active ingredient, cells of a microorganism belonging to the genus Bifidobacterium or Streptococcus. 2. The lipid peroxidation inhibitor according to claim 1, wherein the microorganism belonging to the genus Streptococcus is Streptococcus thermophilus. 3. The microorganism belonging to the genus Bifidobacterium is Bifidobacterium bifidum.
The lipid peroxidation inhibitor according to the above. 4. The lipid peroxidation inhibitor according to claim 1, wherein the microorganism belonging to the genus Bifidobacterium or Streptococcus is Bifidobacterium bifidum YIT 4007 or Streptococcus thermophilus YIT 2001. 5. The lipid peroxidation inhibitor according to claim 1, wherein the lipid peroxidation is induced by iron ions. 6. A preventive / therapeutic agent for intestinal mucosal injury containing the lipid peroxidation inhibitor according to claim 1. 7. A food or drink comprising the lipid peroxidation inhibitor according to claim 1.