KR100691839B1 - Method for increasing bile acid activating enzyme through coculture of Bifidobacterium bipidum and bile acid activating enzyme increasing agent using same - Google Patents

Abstract

The present invention provides a method of increasing bile acid activating enzyme production by co-culturing two or more species selected from the group consisting of Bifidobacterium bipiduum and two or more species selected from the group consisting of Bifidobacterium bipiduum. It relates to a bile acid activating enzyme production increasing agent,

More specifically, a method of increasing the bile acid activating enzyme production by co-culturing Bifidobacterium bifidum species, and comprising at least two species selected from the group consisting of Bifidobacterium bifidum and increasing the production of bile acid activating enzymes or foods It is about. Bile acid activating enzyme increasing agent comprising two or more of the Bifidobacterium bipiduum of the present invention can be used in various lactic acid bacteria products such as fermented milk, health supplements and medicines because of excellent intestinal fixation, acid resistance and bile acid resistance.

Bifidobacterium Bifidum, Bile Acid Activating Enhancer

Description

Method of increasing bile acid activating enzyme through co-culture of Bifidobacterium bipidum and the bile acid activating enzyme increasing agent using the same bibibacterium bifidum, and the composition

Figure 1 shows the effect of bile acid on the pH in each strain and co-culture. Ox: oxal; TCA: Taurocholic acid

Figure 2 shows the conversion efficiency percentage of the starting concentration of bile acids (oxgal and TCA) in a medium containing TYP. The degree of depolymerization is expressed as a percentage of the original concentration of bile acid in the medium.

FIG. 3 shows the percentage of bile acid conjugated by HPLC analysis disappeared into decondensed bile acid. Bb: B. bifidum ; CA: cholic acid; TCA: taurocholic acid

4 shows the effect of several bile acids on plate count as well as growth (OD 650 nm). The toxicity of bile acids at 32 hours of B. bifidum (NRRL 1976, NRRL 1917, KCCM 12096) and combinations thereof (co-culture) in TYP medium added with 0.5% taurocholic acid and oxgal.

The present invention includes a method for increasing bile acid hydrolase through coculture of Bifidobacterium bipidum or bifidobacterium co-culturing, including two or more Bifidobacterium bipidum strains. Activating enzyme increasing agent or a food composition comprising the same.

In order for lactic acid bacteria to have a useful effect as probiotics, they must be able to reach the intestines and proliferate. To this end, in addition to acid resistance and bile acid resistance, it should be excellent in intestinal fixation. Existing lactic acid strains have been mainly researched on acid resistance, bile acid resistance and adhesion ability. 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 function. .

High blood cholesterol is today recognized as a risk factor for cardiovascular diseases, including major health care problems. Epidemic pathological studies have demonstrated, with almost no exception, that those who consume large amounts of unsaturated fat and cholesterol have relatively high serum cholesterol levels and a high mortality rate from coronary heart disease. While other factors also recognize that they may contribute to the progression of cardiovascular disease, there has been a causal relationship between serum cholesterol levels and coronary disease and coronary mortality. Hypercholesterolemia can cause undesirable cholesterol levels in the circulatory system. Accumulation in parts (arteriosclerosis) or soft tissues (yellowing).

HDL (high density lipoprotein) is synthesized in the liver and small intestine, circulates through the blood, and collects the extra cholesterol in the cell and moves it to the liver. HDL carries cholesterol from other tissues to the liver. Therefore, it can be thought that a lot of HDL will remove a lot of cholesterol from blood vessels.

Low density lipoprotein (LDL) is the most dangerous lipoprotein that carries the highest amount of cholesterol and also supplies cholesterol to peripheral cells but causes coronary sinus sclerosis and is obese. Cholesterol, which is produced mainly in the liver, is transported to other tissues such as blood vessels in the form of LDL. There is a lot of cholesterol in the arteriosclerosis of blood vessels. Therefore, a lot of LDL can accumulate a lot of cholesterol in the blood vessels can promote atherosclerosis. Therefore, in general, LDL is described as 'bad cholesterol' that causes arteriosclerosis, and HDL is described as 'good cholesterol' that prevents arteriosclerosis.

Enterero, according to a report by Maghor-Larsen et al. (Agerholm-Larsen L, Raben, Haulrik N, Hansen AS, Manders M, Astrup A., Eur J Clin Nutr. 2000 Apr; 54 (4): 288-97) Yogurt fermented with Enterococcus faecium and two Streptococcus thermophilus have been reported to reduce the amount of LDL-cholesterol. According to a study by Anderson, James W. and SE Gilliland, Journal of American College of Nutrition, Vol. 18, No. 1, 43-50 (1999), Lactobacillus esidophilus isolated from humans ( Fermented milk containing L. acidophilus lowers LDL cholesterol and reports that L. acidophilus, which reduces cholesterol, can reduce the risk of coronary artery disease by 6-10%.

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 you have a lot of conjugated bile acids, the amount of cholesterol absorbed into the blood increases, so the concentration of cholesterol in the blood increases. However, deconjugated bile acid has low solubility and acts to decrease the solubility of cholesterol. Therefore, strains with bile acid hydrolase (BSH) decomplexing conjugated bile acids lower lipid solubility and lower cholesterol absorption in the small intestine. Bifidobacterium is an important part of human intestinal bacterial populations, and Bifidobacterium is mainly added to fermented dairy products because of its health-promoting effect. The most important metabolism of Bifidobacterium is the deconjugation of bile acids that occur naturally in the human intestine. Bile salt hydrolase (BSH) is an important enzyme involved in bile acid depolymerization during the enterohepatic circulation. This bile activator has been found in several lactic acid bacteria, suggesting that bile activator activity is necessary for the selection of probiotic strains with cholesterol-lowering properties. Strains that could not be depolymerized could not remove cholesterol from the culture to a significant extent.

High blood and dietary cholesterol levels are major risk factors for coronary heart disease and are also major factors for colorectal cancer.

Therefore, it is urgent to develop a drug or food using the strain having a cholesterol lowering function for the Korean constitution without side effects.

Accordingly, the present inventors have found that co-culture increases bile acid activating enzymes in the intestine, and uses the increasing agent according to the present invention to increase the bile acid activating enzyme containing two or more strains in fermented milk, dietary supplements and medicines. The present invention has been completed by revealing that it can be.

SUMMARY OF THE INVENTION An object of the present invention is a method of co-culturing Bifidobacterium bipidum strain which increases bile acid activating enzyme, and a food composition comprising the same or an increase agent for increasing bile acid activating enzyme in the intestine by containing two or more strains as an active ingredient. To provide.

In order to achieve the above object, the present invention provides a method for increasing bile acid activator production through co-culturing two or more species selected from the group consisting of Bifidobacterium bipiduum.

The present invention also provides a bile acid activating enzyme production-increasing agent, wherein coculture is induced, including two or more species selected from the group consisting of Bifidobacterium bipidum.

In addition, the present invention provides a food for increasing the production of bile acid activating enzymes, including co-induction, including two or more species selected from the group consisting of Bifidobacterium bipidum.

Hereinafter, the present invention will be described in detail.

The present invention is to use the Bifidobacterium strain as a strain having excellent intestinal fixability, acid resistance and bile acid resistance, blood cholesterol lowering ability, the strain is preferably Bifidobacterium bipiduum.

The origin and types of the three species used in the present invention are shown in Table 1. NRRL-1976 and NRRL-1917 were provided by the ARS Culture Collection (NRRL) in the United States, and KCCM 12096 was provided by the Korean spawn association. The storage culture was maintained at -70 ° C. The bacteria were incubated three times in the appropriate medium before being used for testing in succession. Lactic acid bacteria were grown in TYP medium (16 g of bactodripton / liter, 16 g of yeast extract / liter, 16 g of marine salt / liter [pH 7.5]).

The present invention provides a method for increasing the activity of bile acid activating enzymes through coculture in the intestine, including two or more of the Bifidobacterium bifidum strains.

In another aspect, the present invention provides a co-culture induced, bile acid active enzyme production increasing agent including two or more species selected from the group consisting of Bifidobacterium.

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 can be used as a prophylactic or therapeutic agent for diseases related to cholesterol by lowering cholesterol, and can be particularly useful for the prevention or treatment of obesity, diabetes, hypertension and heart disease.

In general, Bifidobacterium bipidum can be administered orally during clinical administration and can be used in the form of general pharmaceutical preparations. Bifidobacterium Bifidum strains can be administered in various oral dosage forms during actual clinical administration, and when formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents and surfactants that are commonly used Are prepared using. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations include at least one excipient such as starch, calcium carbonate, Bifidobacterium bipidum strains. Sucrose (Sucrose) 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. .

Bifidobacterium bifidum strain is a non-toxic stable strain, the effective dose is 1 x 10 ⁸ to 40 x 10 ⁸ bacteria / kg, preferably 10 x 10 ⁸ to 20 x 10 ⁸ bacteria / kg, a day It may be administered 1-3 times.

In addition, the present invention is co-culture is induced, including two or more species selected from the group consisting of Bifidobacterium bipidu, to provide a food for increasing the production of bile acid active enzyme. 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 used as a food for increasing the production of bile acid activating enzymes to increase the production of bile acid activating enzymes, including co-culture, including two or more species selected from the group consisting of Bifidobacterium bipidum according to the present invention, the strain is added as it is. It may be used in combination 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 the food to which the strain can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, dairy products including other noodles, gum, ice cream, various soups, drinks, tea, drink, 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 (microbial count) of the Bifidobacterium bifidum strain of the present invention per 1 g (or ml) of the specific food material at the time of preparing the food or beverage. And preferably in an amount of from 1 × 10 ⁸ to 5 × 10 ⁸ . The effective dose of the strain of the present invention can 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 certain that it can be used in an amount above the above range.

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.

Test strain and culture conditions

The origin and types of the three species used in the present invention are shown in Table 1. NRRL-1976 and NRRL-1917 were provided by the ARS Culture Collection (NRRL) in the United States, and KCCM 12096 was provided by the Korean spawn association. The storage culture was maintained at -70 ° C. The bacteria were incubated three times in the appropriate medium before being used for testing in succession. Lactic acid bacteria were grown in TYP medium (16 g of bactodripton / liter, 16 g of yeast extract / liter, 16 g of marine salt / liter [pH 7.5]). 1% of the inoculum was used and the culturing was carried out at 37 ° C. for 24 hours in anaerobic conditions (10% H ₂ , 10% CO ₂ and 80% N ₂ ). Inoculation cultures of each strain were taken at the end of the exponential growth phase with a cell concentration of about 10 ⁹ CFU / ml and an absorbance of 2.00 at 650 nm. Co-culturing took 1 ml of each cell suspension and absorbance was mixed at about 650 nm at about 2.00.

Quantitative analysis of bile acid activator titers

Qualitative bile acid activator agar plate testing was performed in TYP medium. NRRL-1976, NRRL-1917, KCCM 12096 and their cocultures were incubated in TYP agar plates containing 0.5% sodium taurocholic acid and oxgall, respectively. Oxal was used in the medium to create conditions similar to the human intestinal environment, and taurocholic acid was used as a source of incorporated bile. Incubation was measured by placing sterile filter discs impregnated in the overnight culture of the test strain on TYP agar plates. The dish was incubated anaerobicly at 37 ° C. for 72 hours, after which the diameter of the precipitation zone was measured. No addition of TYP agar plate was used as a control. Each strain was tested three times, and the strain showing the largest precipitation area was selected.

As shown in Table 1, B. bifidum strain 1976 and co-culture (NRRL 1976, KCCM 12096 co-culture of NRRL 1917) showed the most precipitation area, showing the greatest desaturation effect of TCA and Oxgal. Precipitation and growth zones around the sterile filter paper were measured in mm and were considered measurements of the amount of enzyme secretion and depolymerization of each strain. When co-cultured, bile salt hydrolase was produced in an amount of 3 to 4 times greater than that of each strain.

Determination of Bile Acid Activase Titer by HPLC

The titer of bile acid activating enzyme was measured by HPLC method. To recover the bile acid from the TYP broth culture, cells were removed from 10 ml of sample via centrifugation (10,000 g for 10 min at 5 ° C.). After filtration through a 0.45-mm polysulfone filter, 8 ml of 0.2 M NaOH in 0.9% NaCL was added to 1 ml of filtrate and mixed. The solution was passed through a Sep-Paka C-18 cartridge, which was washed once with 10 ml of water, once with 10% acetone, and again with 10 ml of water. The conjugated or free bile acid was eluted from the cartridge using 5 ml of methanol. The eluate with methanol was evaporated and dried at 60 ° C. under nitrogen gas. The residue was dissolved in 1 ml of mobile phase and filtered analytically through 0.45-mm polysulfone filter paper. Titers of bile acid activators were determined based on the disappearance of taurocholic acid and oxal or both from the medium.

TLC method

Cells in the initial stationary phase were harvested and washed twice with 0.9% NaCl, and the absorbance was adjusted to 10 at 650 nm. 0.5% human bile acid mixture in this cell suspension; 10 μl of 10% taurocholic acid, 10% deoxycholic acid, 5% bile acid and oxgal were added. Samples were taken at 0 hours and 60 minutes. 100 microliters of the sample were extracted with the same amount of ethyl acetate and centrifuged to separate phases. Two microliters on ethyl acetate were loaded on a high performance TLC plate and developed with benzene: isopropanol: acetic acid (30: 1: 30). The plate was dried after development, sprayed with 5% sulfuric acid in a 95% methanol solution and then baked in an oven at 120 ° C. for 5 minutes. The activity of each strain was determined by the degree of spot of the reactant on the high performance TLC plate.

Depolymerization Analysis of Bile Acids During Growth

Prepare B. bifidum in TYP broth (100 ml) to which 0.5 mg / 100 ml sodium taurocholic acid and oxgal are added, respectively. Freshly prepared combinations of these three strains, B. bifidum NRRL-1976, NRRL-1917 and KCCM-12096, were inoculated in 1% medium and mixed for 1 minute. Aseptically remove 10 ml serially from each incubation and transfer to a test tube with a sterile screw-plug. And incubate at 37 ° C. for 24, 32 and 48 hours. Analyzed 24, 32 and 48 hours. Growth is measured by plate count on TYP agar. Samples are also measured for pH, OD (650 nm) and bile acids.

Analysis of the Toxicity of Contained Bile Acids

Each B. bifidum (NRRL-1976, NRRL-1917, KCCM 12096, and their cocultures) was tested for their ability to combat the toxicity of the combined bile acids, TCA, and oxal. A standing culture inoculum (1%) was added to the TYP culture broth with 0.5% TCA and Oxgal respectively. Samples were harvested and analyzed after 0, 24, 48 and 72 hours at 37 ° C anaerobic conditions. Diluted portions were plated on TYP agar plates and incubated for 0, 24, 48, 72 hours at 37 ° C. in anaerobic conditions and viable counts were measured.

As shown in Table 2, B. bifidum 12096 was most sensitive to bile acids and grew less than coculture and each other strain.

Comparison of Cultures on Bile Acid Resistance

Each culture (including coculture) was compared for viability in TYP medium with 0.5% bile acids (TCA and Oxgal). Anaerobic conditions were incubated at 37 ° C. for 0, 24, 48 and 72 hours, and the effect of each bile phase in each culture was measured by increasing or decreasing the absorbance at 650 nm.

As shown in Table 2, B. bifidum (12096) was the most sensitive to bile acids and grew less than B. bifidum 1976 and 1917. The coculture of this bacterial strain showed significantly higher resistance to bile acids.

Quantitative Measurement of Cholesterol

Determination of cholesterol concentration adsorbed to the cells was performed by slightly modifying the method of Rudel and Morris (Rudel, L.L., & Morris, M.D. (1973) .Journal of Lipid research 14, 364-366). 3 ml of 95% ethanol and 2 ml of 50% potassium hydroxide were added to 1 ml of the sample solution. After mixing the contents of the test tube and heated in a 60 ℃ constant temperature water bath for 10 minutes. After cooling, 5 ml of hexane was added to each test tube, and the mixture was sufficiently mixed. Then, 1 ml of distillation was added and the phases were allowed to separate at room temperature. A fraction of 3 ml of the hexane layer was transferred to a clean test tube and the hexane was evaporated under nitrogen gas. 4 ml of O-phthalaldehyde was put in each test tube, waited 10 minutes at room temperature, and then 2 ml of saturated sulfuric acid was added, and after 10 minutes, absorbance was measured at 570 nm. Absorbance was compared to the absorbance standard of cholesterol.

Table 1 shows the results of culturing in TYP medium containing cholesterol and taucolic acid. These results show that TCA is a necessary condition for removing cholesterol to a significant extent. In taucolic acid, there was no significant difference between the strains, but in coculture, there was a big difference. In B. bifidum 1976, 1917 and 12096, and their co-culture, the rate of cholesterol removal after 24 hours was 55, 43, 45, and 65% (co-culture), respectively, and after 32 hours, 60, 45, 50, respectively. And 80% (co-culture). Some cholesterol was redissolved in wash buffer at 29, 28, 25, and 35%, respectively, and remained intact (20, 19, 12, and 29%, respectively) after 24 hours in a significant amount of cholesteroid cells. From this, it can be seen that the removal of cholesterol from the culture solution is eliminated by coprecipitation of depolymerized bile acid and cholesterol. Precipitation of cholesterol appeared to precipitate due to the release of the moiety of cholic acid from taucolic acid due to the activity of the bile acid activating enzyme of the cell (see FIGS. 1 and 2). Taucolic acid was the most efficient bile acid in B.bifidum .

To determine the depolymerization rate of bile acids was analyzed using HPLC (see Figures 1 and 2). Sodium taucholic acid was depolymerized by all strains, strain 1976 in a plate assay test of tatucolic sodium for 72 hours of strain. 81%, 71% for strain 1917, 65% for strain 12096, but 95-98% for the co-culture. Oxgal hydrolase activity for strain 1976 was 78.4%, 58.8% for strain 1917, and 74.8% for strain 12096. On the other hand, in the co-culture, the oxal hydrolase activity was 85%. In vitro elimination of cholesterol has been implicated in the bile acid activating enzymes of cells and appears to secrete depolymerized bile acids which cause precipitation of cholesterol.

Table 1 shows the percentage of cholesterol measured in the supernatant, wash, and cells. All experiments were performed in triplicates and at least two cases were performed. Mean ± standard deviation was indicated and growing cells were incubated for 24 hours in MRS medium containing 0.1 mg / ml cholesterol and 5 mg / ml taurocholic acid or 5 mg / ml.

Table 2 shows the respective strains in TYP medium to which 0.5 g / 100 ml bile acid (B), taurocholic acid (TCA), collic acid (CA), and oxal (OX) were added to determine the toxicity of the bile acid added. Co-culturing B. bifidium shows survival and mean (± standard deviation).

Table 3 shows the plate test analysis whether Bifidobacterium exhibits TCA hydrolase and oxal hydrolase activity.

[Table 1] Percentage of cholesterol measured in supernatant, washing solution and cells

The amount of cholesterol in the different fractions was expressed as a percentage of the starting concentration in the medium.

† Cholesterol solution and bile acid (oxgal or taurocholic acid) were challenged without cells, after which the amount of cholesterol in different fractions was measured.

[Table 2] 0.5g / 100ml bile acid (B), taurocholic acid (TCA), cholic acid (CA), Oxal (OX) added to each strain in the TYP medium to determine the toxicity of the added bile acid And average survival of co-culturing B. bifidum

Table 3 Plate Test Analysis of Bifidobacterium Showing TCA Hydrolase and Oxgal Hydrolase Activity

Dp: diameter of the area to be precipitated; g: indicate growth; p: has TCA hydrolase activity; Each value represents precipitation of cholic acid (defoamer activity; obtained by observing the growth of bacterial populations for 0, 24, 32, 48 hours in TYP medium containing 0.5% TCA and oxal) . Negative analysis showing no TCA-taurocholic acid (contained bile acid), oxgal- (dehydrogenated bile acid) precipitation.

As discussed above, coculture of a species selected from the group consisting of Bifidobacterium bipidu of the present invention increased bile acid activator production. By depolymerization of cholesterol by the bile acid activating enzyme, cholesterol absorption was prevented, and blood cholesterol increase was greatly reduced by food intake. It can be usefully used in fermented milk, health supplements and pharmaceuticals, etc. characterized by a lowering effect.

Claims (6)

Bifidobacterium Bifidum NRRL-1976, NRRL-1917 and KCCM-12096 co-culturing two (2) species selected from the group consisting of increased bile acid activating enzyme production. delete Bifidobacterium bipiduum A bile acid activating enzyme production increasing agent for allowing bifidobacterium bipiduum to be co-cultured, including two (2) species selected from the group consisting of NRRL-1976, NRRL-1917 and KCCM-12096. Bifidobacterium Bifidum comprising a bile acid activating enzyme production increasing agent that allows Bifidobacterium Bifidum to be co-cultured, including two (2) species selected from the group consisting of NRRL-1976, NRRL-1917 and KCCM-12096. Capsules. Bifidobacterium Bifidum NRRL-1976, NRRL-1917 and KCCM-12096 Food for increasing the production of bile acid activating enzymes to allow the Bifidobacterium Bidop co-culture, including two (2) species selected from the group consisting of. 6. The food of claim 5, wherein the food is selected from the group consisting of fermented milk, yoghurt, beverages, milk drinks, food additives, and dietary supplements.

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