KR100930251B1 - Bifidobacterium longum having the ability of reducing cholesterol in blood - Google Patents

Abstract

PURPOSE: A novel Bifidobacterium longum strain is provided to suppress activity of harmful enzyme in intestine and reduces cholesterol in blood. CONSTITUTION: A Bifidobacterium longum SPM1207 strain(KCTC 11506 BP) enhances HDL(High Density Lipoprotein) level and reduces LDL(Low Density Lipoprotein) level and total cholesterol level. An anti-cholesterol agent contains the Bifidobacterium longum SPM1207 strain as an active ingredient.

Description

Bifidobacterium longgum strain having blood cholesterol lowering ability {BIFIDOBACTERIUM LONGUM HAVING THE ABILITY OF REDUCING CHOLESTEROL IN BLOOD}

The present invention relates to a novel Bifidobacterium longum ( Bifidobacterium longum ) strain having a blood cholesterol lowering ability, and more specifically, high density lipoprotein (HDL) levels are increased, total cholesterol levels and low density lipoprotein ( Low Density Lipoprotein (LDL) levels relate to novel Bifidobacterium longgum strains that lower and cholesterol lowering agents or foods containing the strains.

Lactic acid bacteria (lactic acid bacteria) is a bacterium that breaks down carbohydrates and uses it to make lactic acid. Lactic acid bacteria can be divided into five, divided into Streptococcus (Streptococcus), Lactobacillus (Lactobacillus), Lou Pocono Stark (Leuconostoc), bifidobacteria bacteria (Bifidobacteria), and Peddie Oh Caucus (Pediococcus). Microorganisms of Streptococcus, a Streptococcus, are homozygous and are known to inhibit rot and pathogens by fermenting milk to produce lactic acid. The microorganisms of the genus Lactobacillus are lactic acid bacilli that are homozygous or heterozygous, and are commonly found in the fermentation process of dairy products or vegetables, and the microorganisms of the genus ruconosstock, which are heterozygous, are mainly involved in fermentation of vegetables. In addition, Bifidobacteria genus microorganisms are organized anaerobic bacteria that do not grow in the presence of oxygen, ferment sugar to produce L (+) type lactic acid that can be usefully metabolized in infants. Finally, the Pediococcus microorganism is a homozygous fermentation in the form of Lactobacillus, which is present in kimchi or pickled foods and is involved in meat fermentation such as sausage.

Among them, the most lactic acid bacteria in the human intestine are bifidobacteria, which are organized anaerobic lactic acid bacteria, which are known to exist about 100 to 1,000 times more than the anaerobic lactic acid bacteria such as Lactobacillus and Streptococcus. Since bifidus bacteria exist as the most dominant bacteria in the intestines of breastfeeding infants, infants prevent bacterial diarrhea by preventing intestinal proliferation of harmful bacteria from the outside such as Escherichia coli and have the ability to prevent various kinds of bacterial diseases. It is known.

The beneficial health-promoting effects of bifidus bacteria have been reported in various physiological activities such as resistance to infection, anti-cancer and anti-tumor effects, lowering blood cholesterol and intestinal effects (Hentges, DJ 1983. In: Human intestinal microflora in health). and Academic Press.pp. 241-261; Hosono, A., R. Wardardo, and H. Otani. 1990. Inhibitory effects of lactic acid bacteria from fermented milk on the mutagenicities of volatile nitrosoamines.Agricultural and Biological Chemistry. 54 (7): 1639-1643). Recently, as research on lactic acid bacteria has become more active, there is a growing interest in the special functions of lactic acid bacteria such as anticancer effect, immune activity effect, inhibitory effect on certain harmful bacteria (E. coli, Helicobacter pylori, etc.), blood pressure lowering effect and blood cholesterol lowering ability.

In relation to the blood cholesterol lowering ability, a high concentration of blood cholesterol in the body can lead to death and cause cardiovascular diseases such as heart attack and arteriosclerosis. The most common cause of death among Koreans is cerebrovascular disease, traffic accidents, and cancer. Cholesterol is a waxy substance found in the body's bloodstream and lipids in cells. Cholesterol performs the necessary body functions, but too high levels of cholesterol in the blood can be harmful to human health because it increases the risk of cardiovascular disease.

High cholesterol generally means that the total blood cholesterol level in humans exceeds 240 mg / dl or low density lipoprotein (LDL) exceeds 160 mg / dl. When high levels of cholesterol in the blood are reached, cholesterol can be deposited in the arterial walls because cholesterol is not dissolved in the blood. Cholesterol must be transported to or from cells by specific carriers called low density lipoproteins (LDL) and high density lipoproteins (HDL). HDL cholesterol is considered benign cholesterol (good cholesterol) because it removes cholesterol from the blood vessel walls and transports it to the liver to make it digestible. LDL cholesterol is called malignant cholesterol (bad cholesterol) because it carries cholesterol in the blood and accumulates in the blood vessel walls and accelerates atherosclerosis, so the lower this level in the blood, the better.

The mechanism of lowering cholesterol absorption by lactic acid bacteria can be explained in three ways. The first is lactic acid bacteria adsorbed and excreted cholesterol as the intestines settle and proliferate. The second is lactic acid bacteria adsorb and excrete bile acids. In this case, cholesterol is a precursor of bile acid, and thus, cholesterol is used to compensate for the lack of bile acid, which may result in a decrease in intestinal cholesterol concentration. The third mechanism is the action of lactic acid bacteria to depolymerize bile acid. The free bile acid produced by the liver combines with taurine or glycine in the small intestine to become conjugated bile acid. Polybile bile acids have high lipid solubility and facilitate absorption of lipids such as cholesterol in the small intestine. Therefore, if the amount of conjugated bile acid is high, the amount of cholesterol absorbed into the blood increases and the concentration of cholesterol in the blood increases. However, deconjugated bile acid has a low lipid solubility, thereby lowering blood transduction of lipids such as cholesterol. Therefore, strains having bile acid hydrolase decomplexing conjugated bile acids may lower lipid solubility, thereby lowering the absorption of cholesterol in the small intestine. Correlation between side effects and carcinogenic components related to cholesterol and bile acid metabolism by cholesterol-lowering lactic acid strains is not clear yet, and further studies should be conducted through animal or human studies. Therefore, it is urgent to develop a drug or food using the strain having a cholesterol lowering function for the Korean constitution without any side effects.

The inventors of the present invention, while continuing to research to develop a novel lactic acid bacteria with excellent cholesterol-lowering ability without side effects, isolating new Bifidobacterium microorganisms from healthy Korean servings, high density lipoprotein (HDL) Increases cholesterol levels and lowers density of low density lipoprotein (LDL), while inhibiting the activity of intestinal harmful enzymes, preventing colon cancer, increasing the number of lactic acid bacteria, the beneficial bacteria in the intestines, and water content in feces The present invention was completed by confirming that it has the effect of enhancing the effect of controlling the mouth.

It is an object of the present invention to provide a novel Bifidobacterium longgum microorganism having excellent activity of lowering cholesterol in the blood and inhibiting the activity of harmful intestinal enzymes and increasing fecal moisture content while increasing the number of intestinal lactic acid bacteria.

In order to achieve the above object, the present invention increases the high density lipoprotein (HDL) level, lowers the total cholesterol level and low density lipoprotein (LDL) level, while inhibiting the activity of harmful intestinal enzymes. It provides a Bifidobacterium long gum strain having the activity of increasing the fecal moisture content.

The present invention also provides a cholesterol lowering agent containing the strain as an active ingredient.

The present invention also provides a food for lowering cholesterol containing the strain as an active ingredient.

Hereinafter, the present invention will be described in detail.

The present invention increases high density lipoprotein (HDL) levels, decreases total cholesterol and low density lipoprotein (LDL) levels, inhibits the activity of intestinal harmful enzymes, and increases fecal moisture content. Provided are Bifidobacterium longgum strains having activity.

Physiological biochemical properties of the Bifidobacterium longgum strain of the present invention are shown in Table 1 below. In addition, the Bifidobacterium longgum strain of the present invention has a 16S rRNA gene comprising a nucleotide sequence of 1,520 bp shown in FIG.

Sugar utilization of Bifidobacterium longum SPM1207 Sugar B. longum SPM1207 Cellobiose - D-Ribose - Galactose + Gluconate - Fructose - Inuline + Lactose + L-Srabinose + Maltose + Mannitol - Mannose - Melibiose + Melezitose + Raffinose + Saccharose + Salicine - Sorbitol - Starch - Trehalose - Xylose +

In the preferred embodiment of the present invention, the high density lipoprotein (HDL) level is increased, the total cholesterol level and low density lipoprotein (LDL) level are lowered, while inhibiting the activity of intestinal harmful enzymes, An excellent strain that increases the intestinal lactic acid bacteria count and increases the water content of feces is named 'Bifidobacterium long gum SPM1207', and it was deposited on May 12, 2009 to the Korea Microorganism Conservation Center (Accession No .: KCTC 11506BP)

When the Bifidobacterium longgum strain of the present invention is cultured in a cholesterol-containing medium, cholesterol is adsorbed to the cells and the amount of cholesterol remaining in the culture filtrate is drastically reduced (see Table 3 below). In addition, in the present invention, after the Bifidobacterium ronggeom strain of the present invention maridang in rats fed during ^{0.2 ㎖ (10 8 ~ 10 9} cfu / ㎖) 2 ju each day, total cholesterol and whole blood processing (TC: Total Cholesterol) levels The high density lipoprotein (HDL) level, and low density lipoprotein (LDL) level were analyzed to observe the effect of the strain of the present invention. As a result, when the Bifidobacterium longgum SPM1207 strain of the present invention is administered to rats with cholesterol, the Bifidobacterium longgum KCTC 3352, KCTC 3128, Lactobacillus ash, which is known to not administer the strain of the present invention or to reduce cholesterol, The amount of cholesterol in the blood is drastically reduced compared to the Dophilus and Frantarum strains (see Table 3 below).

In addition, the Bifidobacterium longgum strain of the present invention decreases the activity of harmful enteric enzymes in the intestine and increases the number of intestinal lactic acid bacteria, thereby reducing blood cholesterol without side effects. To the present invention, the sample collection defecation ronggeom Bifidobacterium strains of the invention in rats and maridang benefit for ^{0.2 ㎖ (10 8 ~ 10 9} cfu / ㎖) 2 ju every day, every week, β- glucosidase in the sample As a result of observing the effects of the Bifidobacterium longgum strain of the present invention by measuring enzymatic activities such as sidase, β-glucuronidase, tryptophanase, alkaline phosphatase, and urease, the Bifidobacterium longgum of the present invention SPM1207 lactic acid bacteria also inhibited indole-producing tryptophanase enzyme activity known to cause bladder cancer and colorectal cancer, and also inhibited urease enzyme activity. In addition, the strains of the present invention increase the number of intestinal lactic acid bacteria to promote the production of organic acids, it was observed to increase the water content in the feces, it can be seen that the Bifidobacterium long gum strain of the present invention enhances the stool effect . In addition, as a result, it could be observed that in the case of the mice administered the strains of the present invention, the weight was increased in comparison with the mice not.

The strain of the present invention inhibits the activity of harmful enzymes such as tryptopanase and urease in the intestine, increases the number of lactic acid bacteria for the total number of bacteria in the large intestine, and increases the water content in the feces, thereby preventing the bleeding of the present invention. Terium longgum strains have an effect of lowering blood cholesterol content.

The present invention also provides a cholesterol lowering agent containing the Bifidobacterium long gum strain as an active ingredient. Stamler et al. (Arch. Pathol. Lab. Med., 1988, 112: 1032-1040) reported that decreased blood cholesterol can prevent or treat heart disease. Sindelka et al. (Physiol. Res., 2002, 51: 85-) 91) reported that blood cholesterol was also associated with obesity, diabetes and hypertension. Therefore, the cholesterol lowering agent of the present invention may be used as a prophylactic or therapeutic agent for diseases related to cholesterol by lowering cholesterol, and particularly useful for the prevention or treatment of obesity, diabetes, hypertension and heart disease.

The Bifidobacterium longgum strain of the present invention can be administered orally during clinical administration and can be used in the form of a general pharmaceutical formulation. That is, the Bifidobacterium longgum strain of the present invention may be administered in various oral dosage forms during actual clinical administration. When formulated, Bifidobacterium longgum strains such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, and the like, which are commonly used Prepared using diluents or excipients. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like. Such solid preparations include at least one excipient such as starch, calcium carbonate, Bifidobacterium long gum strain, Sucrose (Lucose) or lactose (Lactose), gelatin and the like are mixed and prepared. In addition to simple excipients, lubricants such as magnesium styrate talc are also used. Oral liquid preparations include suspending agents, liquid solutions, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. .

The Bifidobacterium longgum strain of the present invention is a safe non-toxic strain, and an effective dose is 2 × 10 ⁸ to 2 × 10 ⁹ cfu / kg and may be administered 1 to 3 times a day.

In addition, the present invention provides a food for lowering cholesterol containing the Bifidobacterium long gum strain as an active ingredient. Cholesterol-lowering foods of the present invention can be used as a prophylactic or therapeutic agent for cholesterol-related diseases, in particular can be useful for the prevention or treatment of obesity, diabetes, hypertension and heart disease.

When the Bifidobacterium long gum strain of the present invention is used as a food, the strain may be added as it is or used with other food or food ingredients, and may be appropriately used according to a conventional method. There is no particular limitation on the kind of food. Examples of foods to which the strain can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, dairy products including other noodles, gums, ice cream, various soups, drinks, tea, drinks, Alcoholic beverages and vitamin complexes, and the like, and are preferably prepared with fermented milk, yoghurt, cheese, milk drink or milk powder, and more preferably fermented milk, yoghurt, beverage, milk powder, food additives or health supplements.

The mixed amount of the active ingredient can be suitably determined depending on the purpose of use (prevention, health or therapeutic treatment). In general, 1 x 10 ⁷ to 10 x 10 ⁸ (bacteria) of the Bifidobacterium long gum strain of the present invention per 1 g (or ml) of the specific food material is added at the time of preparing the food or beverage. And preferably in an amount of 1 × 10 ⁸ to 5 × 10 ⁸ . The effective dose of the strain of the present invention may be used in accordance with the effective dose of the pharmaceutical composition, but may be less than the above range in the case of long-term intake for the purpose of health and hygiene or health control, Since there is no problem in terms of safety, it is evident that it can be used in an amount above the above range.

As described above, the Bifidobacterium longgum strain of the present invention decreases total cholesterol and total cholesterol and low density lipoprotein (LDL) levels in the blood, while inhibiting the activity of intestinal harmful enzymes and intestinal total The number of lactic acid bacteria is characterized by increasing activity, and may be usefully used in fermented milk, dietary supplements, and medicines.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Example 1 Isolation and Characterization of Bifidus Strains

<1-1> Collection of feces

The present inventors collected feces from healthy adults of 20 to 30 years old to isolate the Bifidobacteria strains present in the intestines of Koreans.

<1-2> Species Separation and Characterization

We collected 1 g of fecal samples from healthy Korean adults aged 20-30 with normal eating habits into a sterile disposable plastic bag (Seward Co., UK) containing 100 ml of saline. The stool suspension was mixed well using a mixer (Seward Co., UK) and sequentially diluted with physiological saline. 0.1 ml of each diluted 10-fold with physiological saline was taken, and then mixed with N ₂ (90%), H ₂ (5%) and CO ₂ (5%) in a BL agar plate containing 5% sheep serum. The gas-filled anaerobic system (Bactron Anaerobic Chamber, Sheldon Manufacturing Inc., USA) was incubated at 37 ° C. for 48 hours.

Among colonies growing on BL agar plates, light brown, milky white or tan brown colonies with a reddish brown central part of 0.5 to 2.0 mm were selected and cultured in a new General Anaerobic Media (GAM) medium. To a GAM agar slant. The isolated and purified colonies were gram stained and microscopically examined for the presence of predominant bifidus bacteria. All expected Bifidus colonies were incubated for 24 hours at 37 ° C. in 10 ml of GAM broth.

In addition, the F6PPK (fructose-6-phosphokinase, fructose-6-phosphate phosphoketolase) test (Scardovi V, B sgorbati and G Zani, J) shows whether there is F6PPK activity, a unique and important enzymatic activity of Bifidus. Based on Bacteriol, 1971, 106, 1036-1039, it was confirmed that the isolated bacterial species belong to the genus Bifidobacterium. Practical identification of Bifidobacterium at the species level was performed according to Bergey's guidelines (Scardovi V, Bergey's manual of systematic bacteriology, 1986, 1418-1434).

Example 2 Molecular Biology of Selected Bifidobacterium Bacteria-16S rRNA Analysis

Example 2-1 Preparation of RNA

Cells were harvested from 10 ml of bifidus culture and suspended in 0.5 ml of ice-cold TE (pH 7.5) buffer, followed by 0.6 g glass beads (0.1 mm or less), 0.17 ml macaloid clay, 0.5 ml Phenol-chloroform (1: 1), 50 μl 10% SDS were added and vigorously mixed at 4 ° C. for 5 minutes and then centrifuged at 12,000 rpm for 15 minutes.

The supernatant was extracted with phenol-chloroform (1: 1) solution to precipitate RNA for 30 min at -20 ° C with 1/10 fold of 3 M sodium-acetate and 3 folds of 96% ethanol, followed by centrifugation (12,000 rpm, 15 minutes) and the precipitated pellet RNA was washed with 70% ethanol. The pellet RNA was then dried at room temperature and suspended in 50 μl TE pH 7.5 buffer.

Example 2-2 Preparation of DNA for Sequence Analysis

Cells were recovered from 10 ml of bifidus culture incubated overnight, washed with 1 ml TES (20 mM Tris-HCl, 0.1 mM Na2EDTA, 10 mM NaCl) buffer and suspended in 100 μl TES buffer. Lysozyme (Lysozyme) and mutanolysine (mutanolysin) was treated for 15-30 minutes at 37 ℃ and dissolved with SDS (sodium dodecyl sulfate) for 5 minutes at 65 ℃.

Thereafter, the mixture was incubated on ice for 1 hour by adding NaCl, and then centrifuged at 12,000 rpm for 20 minutes, and DNA was extracted with the same amount of chloroform / isoamyl alcohol. Purified DNA was precipitated with 1/9 times 3M sodium acetate solution and 2 times 96% ethanol and washed with 70% ice-cold ethanol (ice-cold ethanol), followed by DNA samples.

Example 2-3 Sequence Analysis

16S rRNA analysis of SPM 1207 was analyzed by the macrogen industry. 16S rRNA gene of SPM1207 is shown in Figure 1, and has a nucleotide sequence of 1,520bp. The similarity between the 16S rRNA gene and the known bacterial 16S rRNA gene sequence was calculated by the Blast similarity search program (National Institute of Biotechnology Information). This sequence showed 99% homology with the 16S rRNA sequence of Bifidobacterium longgum DJO10A.

As a result of identifying isolated strains as in Examples 2-1 and 2-2 with physiological biochemical and molecular biological characteristics, these strains were identified as Bifidobacterium longgum. The inventors named the strain 'Bifidobacterium longgum SPM1207' and deposited it on May 12, 2009 at the Korea Microorganism Conservation Center (Accession No .: KCTC11506BP).

Example 3 In Vitro Cholesterol Lowering Effect Experiment

The present inventors measured the cholesterol lowering efficacy in vitro of the SPM1207 strain of the present invention isolated and identified in Example 2 as a comparative example of the strain already commercially used as shown in Table 2 below.

Each strain of Examples and Comparative Examples was inoculated in MRS medium containing 0.05% L-cysteine.HCl.H ₂ O and water soluble cholesterol (polyoxyethanyl-cholesterol sebacate, Sigma, USA), and cultured at 37 ° C. for 24 hours. The content of cholesterol remaining in the was measured by the method of Rudel and Morris (Rudel Morris, J. Lipid Research, 1973. 14: 364-366).

Types of Strains Used in Examples and Comparative Examples Bacterial strains Source Origin Example Bifidobacterium longum SPM1207 Isolate Human feces Comparative Example 1 Bifidobacterium adolescentis KCTC3352 Commercial Intestine of adult Comparative Example 2 Bifidobacterium longum KCTC3128 Commercial Intestine of adult Comparative Example 3 Lactobacillus acidophilus (LH) CBT Commercial Not available Comparative Example 4 Lactobacillus plantarum KCTC1048 Commercial Not available Comparative Example 5 Lactobacillus plantarum (LP) CBT Commercial Not available

Cholesterol lowering rate is as follows.

Cholesterol lowering rate (%) = (control O.D-test O.D / control O.D) X 100

                       (Test group: Cholesterol O.D of culture filtrate cultured with isolated bacteria;

                        Control group: cholesterol O.D.

In the Bifidobacterium long gum SPM1207 strain and the comparative strain of the present invention, the cholesterol lowering rate was as shown in Table 3 below.

Cholesterol lowering rate Species Residual cholesterol (mg / dl) Cholesterol lowering ratio (%) p value Control MRS brotoh containing cholesterol without lactic acid bacteria 345.0 ± 5.7 - - Example Bifidobacterium longum SPM1207 262.8 ± 1.3 23.8 p <0.05 Comparative Example 1 Bifidobacterium adolescentis KCTC3352 329.0 ± 5.6 4.6 p <0.05 Comparative Example 2 Bifidobacterium longum KCTC3128 303.8 ± 6.0 11.9 p <0.05 Comparative Example 3 Lactobacillus acidophilus (LH) CBT 321.7 ± 1.9 6.7 p <0.05 Comparative Example 4 Lactobacillus plantarum KCTC1048 314.0 ± 4.4 9.0 p <0.05 Comparative Example 5 Lactobacillus plantarum (LP) CBT 296.3 ± 5.0 14.1 p <0.05

p <0.05, showing an effective effect compared to the control

As shown in Table 3, in the case of the Bifidobacterium long gum SPM1207 of the present invention, cholesterol was reduced by 23.8% from 345.0 to 262.8, and Comparative Examples 1 to 5 ( Bifidobacterium), which were known to have a conventional cholesterol lowering effect adolescentis KCTC3352, Bifidobacterium longum KCTC3128, Lactobacillus acidophilus (LH) CBT, Lactobacillus plantarum KCTC1048, Lactobacillus plantarum (LP) CBT) is significantly higher than the effect of up to five times.

Example 4 Blood Cholesterol Lowering Effect

The present inventors conducted the animal test of the strain of the present invention selected in Example 3 to determine the rate of cholesterol concentration reduction in the in vivo environment.

Specifically, the test animals were 5 week-old SD rat males from Central Lab Animal Inc. It was purchased from and used as i) control group fed with high cholesterol feed and physiological saline, ii) group fed with high cholesterol feed and SPM1207 lactobacillus, and iii) general group fed with normal feed. Breeding. The composition of the high cholesterol feed is shown in Table 4. The SPM1207 strain was administered 0.2 ml once a day for 2 weeks at a concentration of 10 ⁸ ~ 10 ⁹ cfu / ml.

Composition of High Cholesterol Feed Ingredients Compositions g kcal Casein (from milk) 200 800 Corn starch 155.036 620 Sucrose 50 200 Dexrose 132 528 Cellulose 50 0 Soybean oil 25 225 Lard 175 1575 Mineral mixture 35 0 Vitamin mixture 10 40 TBHQ 0.014 0 DL-Methionine 0 0 L-Cystine 3 12 Choline bitartrate 2.5 0 Total 838 4,000 Cholesterol 20 g / kg (2%) Cholic acid 5 g / kg (0.5%)

Each group received three weeks of each feed, and during the last two weeks, i) group received normal saline, and ii) group received SPM1207 strain. Three weeks later, blood was collected from the heart of rats to measure blood cholesterol levels, and serum was separated by centrifugation (3500 rpm / 10 min). The separated serum was referred to SamKwang Medical Foundation for analysis.

Blood Cholesterol Level Measurement Index Group i Group ii Group iii p value Total cholesterol (mg / dl) 111.3 ± 5.2 84.8 ± 4.3 55.0 ± 5.5 p <0.05 ^c HDL-cholesterol (mg / dl) 30.0 ± 2.6 30.6 ± 1.5 23.9 ± 1.4 p> 0.05 ^c LDL-choletsterol (mg / dl) 33.3 ± 8.5 23.5 ± 2.3 11.6 ± 1.1 p <0.05

The total cholesterol level was 113.3 mg / dl to 84.8 mg / dl and the LDL-cholesterol level was 33.3 in the group ii) fed the high cholesterol feed and the bifidoron gum SPM1207 of the present invention together. HDL-cholesterol levels slightly increased from 30.0 mg / dl to 30.6 mg / dl, compared to 23.5 mg / dl at mg / dl.

Based on the above results, the lactic acid strain of the present invention or the mixed strains of the present invention seems to be able to exhibit a large cholesterol lowering effect when used as a drug and a functional food.

<Example 5> Intestinal harmful enzyme activity inhibitory effect

In order to determine the effect of the Bifidobacterium longgum SPM1207 of the present invention on the reduction of intestinal harmful enzymes, the activity of the harmful enzymes due to the intestinal bacteria remaining in the feces of rats to which the SPM1207 strain of the present invention was administered was measured. Each week, feces were collected directly from rats, and 0.9 ml of 0.1M phosphate buffer (pH 6.8) containing 0.5% cysteine per 0.1 g of feces was added and suspended. The enzymatic activities of -glucosidase, β-glucuronidase, tryptopanase and urease were measured.

Example 5-1 Determination of β- glucosidase enzyme activity

1 ml of a reaction solution consisting of 0.8 ml of 2 mM p-nitrophenyl-β-D-glucopyranoside and 0.2 ml of suspended enzyme solution (suspended fecal sample) is reacted at 37 ° C for 30 minutes, and then 1 ml of 0.5N NaOH is added to terminate the reaction. . Thereafter, the reaction solution was centrifuged (3,000 rpm / 10 min), and then the supernatant was measured for absorbance at 405 nm.

Example 5-2 Determination of β- glucuronidase Enzyme Activity

1 ml of a reaction solution consisting of 0.8 ml of 2 mM p-nitrophenyl-β-D-glucuronide and 0.2 ml of suspended enzyme solution was reacted at 37 ° C. for 30 minutes, and 1 ml of 0.5N NaOH was added to terminate the reaction. Thereafter, the reaction solution was centrifuged (3,000 rpm / 10 min), and then the supernatant was measured for absorbance at 405 nm.

<Example 5-3> Tryptopanase enzyme activity measurement

0.2 ml of complete reagent solution (0.25 ml of 20 mM tryptophan) and enzyme solution suspended in 100 ml of 0.05 M potassium phosphate buffer containing 2.75 mg of pyridoxal phosphate, 19.6 mg of disodium EDTA dihydrate, 10 mg of bovine serum albumin and pH 7.5 The reaction solution consisting of ㎖ was reacted at 37 ° C. for one hour, and 2 ml of color reagent (ρ-dimethylaminobezaldehyde 14.7 g, 95% ethanol 948 ml, H ₂ SO ₄ 52 ml) was added to terminate the reaction, followed by centrifugation. (3,000 rpm / 10 min) and the supernatant was measured for absorbance at 550 nm.

<Example 5-4> Urease Enzyme Activity

A reaction solution consisting of 0.3 ml of urea substrate solution (solution dissolved in 4 mM urea in 20 mM sodium phosphate buffer) and 0.1 ml of suspended enzyme solution was reacted at 37 ° C. for 30 minutes, and 0.1 ml of 1 N (NH ₄ ) ₂ SO _{4 was} added. The reaction was terminated by addition.

Thereafter, 1 ml of a phenolnitroprusside reagent and 1 ml of an alkaline hypochlorite reagent (NaClO) were added to the reaction solution for 20 minutes at 65 ° C. The reaction solution was centrifuged at 3,000 rpm for 10 minutes, and the supernatant was measured for absorbance at 603 nm.

The results of the above experiments are shown by calculating the percentage inhibition compared to before administration of the Bifidobacterium longgum SPM1207 of the present invention.

As shown in FIG. 2, after administration of the Bifidobacterium longgum of the present invention, the Bifidobacterium longgum SPM1207 lactic acid bacterium of the present invention, an intestinal harmful enzyme, inhibits the activity of tryptophanase enzyme that produces indole, which is known to cause bladder cancer and colorectal cancer. It is understood that the urease enzyme activity is also suppressed.

Example 6 Intestinal Lactic Acid Bacteria Increase Effect

Intestinal lactic acid bacteria were measured for each week for each of groups i), ii) and iii) of Example 4.

As shown in FIG. 3, in the case of group ii) administered Bifidobacterium longgum SPM1207 of the present invention, the intestinal lactic acid bacteria count was significantly increased from 5.3 log ₁₀ cfu / g to 6.9 log ₁₀ cfu / g, and thus, the group administered with cholesterol alone. In contrast, the Bifidobacterium longgum strain of the present invention reaches the intestine through the gastric acid to increase the number of lactic acid bacteria to the total bacterial count and promote the generation of organic acids, resulting in improving the intestinal environment and promoting the effect of mouth pain. It can be seen that.

Example 7 Effect of Increasing Moisture Content in Feces

For each of the groups i), ii) and iii) of Example 4, the water content in the feces was measured for each week.

As shown in FIG. 4, in the case of group ii) administered Bifidobacterium longgum SPM1207 of the present invention, the intestinal water content was significantly increased than before administration so that the intestinal water content was higher than that in the case of administration of cholesterol alone. It can be seen that it improves the environment and enhances the effect of speaking.

<Example 8> Observation of the change in weight according to the administration of Bifidobacterium long gum strain of the present invention

For each of the groups i), ii), and iii) of Example 4, weight changes were specified for each week.

As shown in FIG. 5, in the case of the group ii) administered Bifidobacterium longgum SPM1207 of the present invention together with the cholesterol feed, it was observed that the weight gain was decreased over time compared to the group iii) given only the cholesterol feed.

1 shows the 16S rRNA sequence of Bifidobacterium longgum SP1207 (KCTC 11506 BP) of the present invention.

Figure 2 shows the activity of intestinal harmful enzymes before and after administration of Bifidobacterium longgum SP1207 (KCTC 11506 BP) of the present invention.

(A) Tryptophanase activity,

(B) Urease activity

(C) β-glucosidase activity

(D) β-glucuronidase activity

Figure 3 shows the change in the intestinal lactic acid bacteria count in each group administered Bifidobacterium longgum SP1207 (KCTC 11506 BP) and cholesterol of the present invention.

Figure 4 shows the change in water content in feces for each group administered Bifidobacterium longgum SP1207 (KCTC 11506 BP) and cholesterol of the present invention.

5 shows the change in weight in each group administered Bifidobacterium longgum SP1207 (KCTC 11506 BP) and Bifidobacterium longgum SP1207 (KCTC 11506 BP) and cholesterol of the present invention.

<110> SAHMYOOK UNIVERSITY INDUSTRY-ACADEMIC COOPERATION FOUNDATION <120> BIFIDOBACTERIUM LONGUM HAVING THE ABILITY OF REDUCING CHOLESTEROL          IN BLOOD <130> KPA09008 <140> 10-2009-42497 <141> 2009-05-15 <160> 1 <170> KopatentIn 1.71 <210> 1 <211> 888 <212> DNA <213> Bifidobacterium longum SPM1207 <400> 1 gnnnngnntt tccggnatta ttgggcgtaa gggctcgtag gcggttcgtc gcgtccggtg 60 tgaaagtcca tcgcttaacg gtggatccgc gccgggtacg ggcgggcttg agtgcggtag 120 gggagactgg aattcccggt gtaacggtgg aatgtgtaga tatcgggaag aacaccaatg 180 gcgaaggcag gtctctgggc cgttactgac gctgaggagc gaaagcgtgg ggagcgaaca 240 ggattagata ccctggtagt ccacgccgta aacggtggat gctggatgtg gggcccgttc 300 cacgggttcc gtgtcggagc taacgcgtta agcatcccgc ctggggagta cggccgcaag 360 gctaaaactc aaagaaattg acgggggccc gcacaagcgg cggagcatgc ggattaattc 420 gatgcaacgc gaagaacctt acctgggctt gacatgttcc cgacggtcgt agagatacgg 480 cttcccttcg gggcgggttc acaggtggtg catggtcgtc gtcagctcgt gtcgtgagat 540 gttgggttaa gtcccgcaac gagcgcaacc ctcgccccgt gttgccagcg gattatgccg 600 ggaactcacg ggggaccgcc ggggttaact cggaggaagg tggggatgac gtcagatcat 660 catgcccctt acgtccaggg cttcacgcat gctacaatgg ccggtacaac gggatgcgac 720 gcggcgacgc ggagcggatc tctgaaaacc ggtctcagtt cggatcgcag tctgcaactc 780 gactgcgtga aggcggagtc gctagtaatc gcgaatcagc aacgtcgcgg tgaatgcgtt 840 cccgggcctt gtacacaccg cccgtcaagt catgaaagtg ggcagcac 888  

Claims (7)

In the Bifidobacterium longum SPM1207 strain (KCTC 11506 BP), which is effective in lowering blood cholesterol levels, The Bifidobacterium longum ( Bifidobacterium longum ) SPM1207 strain increases high density lipoprotein (HDL) levels and decreases the total cholesterol resin and low density lipoprotein (LDL) levels of Bifidobacterium Bifidobacterium longum SPM1207 strain (KCTC 11506 BP). delete The method of claim 1, The Bifidobacterium long gum ( Bifidobacterium) longum ) SPM1207 strain is a Bifidobacterium longgum ( Bifidobacterium) which inhibits the activity of tryptophanase and urease enzymes. longum ) SPM1207 strain (KCTC 11506 BP). The method of claim 1, The Bifidobacterium long gum ( Bifidobacterium) longum ) SPM1207 strain is a Bifidobacterium long gum ( Bifidobacterium ) which increases the number of lactic acid bacteria in the intestine longum ) SPM1207 strain (KCTC 11506 BP). The method of claim 1, The Bifidobacterium long gum ( Bifidobacterium) longum) SPM1207 strain of Bifidobacterium would ronggeom (Bifidobacterium to increase the water content of the stool longum ) SPM1207 strain (KCTC 11506 BP). A cholesterol lowering agent containing Bifidobacterium longum SPM1207 strain (KCTC 11506 BP) according to any one of claims 1 and 3 to 5 as an active ingredient. A cholesterol-lowering food comprising the Bifidobacterium long gum SPM1207 strain ( Bifidobacterium longum ) according to any one of claims 1 to 3 to 5 (KCTC 11506 BP) as an active ingredient.

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