JP6381869B2 - Lactic acid bacteria derived from Tsuda turnip which have liver triglyceride reducing effect - Google Patents

Description

  The present invention relates to a lactic acid bacterium having an action of reducing liver neutral fat, and an oral composition containing the lactic acid bacterium or a fermented product thereof, and particularly relates to a food or drink composition or a pharmaceutical composition having an action of reducing liver neutral fat.

  Metabolic syndrome caused by abnormal lipid metabolism is a symptom in which two or more of hypertension, hyperglycemia, and hyperlipidemia are combined with visceral fat obesity, and various preventive measures have been studied. It has not yet been solved. In recent years, the precautionary idea of preventing symptoms by meals is becoming the focus (Non-patent document 1), intestinal regulating action (Non-patent document 2), immunostimulating action (Non-patent document 3), and lipids Probiotics of lactic acid bacteria, which have reports on blood cholesterol lowering action (Non-patent Document 4) and lipid metabolism improving effect (Non-Patent Document 5), are attracting attention.

"General Chart of Medical System Reform" December 1, 2005 Government and Ruling Party Medical Reform Council Yukimasa Higashi, Kazunori Ito, Atsushi Oki, Akihiro Inoue, Kazuhisa Inoue, Manabu Sato, Yoshitsugu Kanno, Lactobacillus gasseri NY0509 and Lactobacillus casei NY1301 Effects of fermented milk drinks on fecal flora in healthy adults, Journal of Food Science and Technology 48 , (2001), 35 M. Kimura, K. Danno, H. Yasui, Immunomodulatory Function and Probiotic Properties of Lactic acid Bacteria Isolation from Mongolian fermented Milk, Bioscience Microflora, 25, (2006), 147-155 R. M. Pigeon, E. P. Cuesta, S. E. Gilliland, Binding of Free Bile acids by Cells of Yogurt Starter Culture Bacteria J. Dairy Sci., 85 (2002), 2705-2710 Tomoko Harada, Maki Tanaka, Kimiko Tsuzuki, Makoto Sugawara, Naoki Takagi, Mitsuru Fukuda, Shokuhin, 57, (2001), 175-179 M. Kimura, K. Hayakawa, H. Sansawa, Involvement of γ-Amino butyric acid (GABA) B Receptors in the Hypotensive Effect of Systemically Administered GABA in Spontaneously Hypertensive Rats, Jpn. J. Phrarmacol., 89, (2002), 388-394 A. M. Abdou, S. Higashiguchi, K. Horie, Mujo Kim, H. Hatta, H. Yokogoshi, Relaxation and immunity enhancement effects of Aminobutyric acid (GABA) administration in humans, Bio Factors., 26, (2006), 201-208 S. Saski, C. Thoda, M. Kim, T. Yokozawa γ-Aminobutyric acid Specifically Inhibits Progression of Tubular Fibrosis and Atrophy in Nephrrectomized Rats Biol. Pharm. Bull., 30, (2007), 687-687 Folch, J., Lees, M. and Stanley, G.H.S. (1957) A simple method for the isolation and purification of total lipides from animal tissues.The journal of Biological Chemistry 226, 497-509 Frank. A. M. Klaver, Roelof van der. Meer, The Assumed Assimilation of Cholesterol by Lactobacilli and Bifidobacterium bifidum Is Due to Their Bile Salt-Deconjugating Activity, Appl. Env. Micro, 59, (1993), 1120-1124 T. D. Tnguyen, J. H. Kang, M. S. Lee, Characterization of Lactobacillus plantarum PH04, a potential prebiotic bacterium with cholesterol-lowering effects, Int. J. Food Microbiology, 113 (2007), 358-361 M. P. Talanto, F. Sesma, A. Pesce de Ruiz Holgado and G.F. deVal dez, Bile salts hydrolase plays a key role on cholesterol removal by Lactobacillus reuteri, Biotechology Letters, l19, (1997), 845-847 H. Kimoto, S. Ohomomo, T. Okamoto, Cholersterol Removal from Media by Lactococci, J. Dairy Sci, 85, (2002), 31382-31388. Derek K. Walker, Stanley E. Gilliland, Relationships Among Bile Tolerance, Bile Salt Deconjugation, and Assimilation of Choresterol by Lactobacillus acidophilus, J. Dairy. Sci, 76, (1993), 956-961 YK Lee, CY Lim, WL Teng, AC Ouwehand, EM Tuomola, S. S alminen, Quantitative Approach in the study of Adhesion of Lactic Acid Bacteria to Intestinal Cells and Their Competition with Enterobacteria.Appl.Environ.Microbiol., 66, ( 2000), 3692-3697

  The present invention relates to a lactic acid bacterium having an action of reducing the neutral fat of the liver, which causes fatty liver, particularly a liver neutral fat reducing action, a γ-aminobutyric acid producing action and a cholesterol lowering action, and a metabolic syndrome An object of the present invention is to provide a lactic acid bacterium that can be used as a probiotic effective in preventing or improving the above. Moreover, this invention aims at providing the oral composition containing the said lactic acid bacteria or its fermented material, for example, a food-drink composition, and a pharmaceutical composition.

In order to solve the above problems, the present inventor focused on Tsuda turnip which is a special product of Matsue City, Shimane Prefecture, and intensively studied.Lactobacillus brevis belonging to Lactobacillus brevis among lactic acid bacteria present in Tsuda turnip, liver Has been found to have an effect of reducing neutral fat. Moreover, in addition to the liver triglyceride reducing effect, the lactic acid bacterium has a blood pressure increase inhibiting effect (Non-patent document 6), a relaxing effect (Non-patent document 7), a kidney function improving effect (Non-patent document 8), and the like. It was confirmed that there was an action to produce γ-aminobutyric acid (GABA), a reported inhibitory neurotransmitter, and an action to lower cholesterol.

  The present invention has been completed based on this finding, and provides the lactic acid bacteria derived from the Tsuda turnip as a new functional material for reducing the neutral fat of the liver. Moreover, the lactic acid bacteria derived from the Tsuda turnip are provided as a new functional material for preventing and improving metabolic syndrome from both aspects of lipid metabolism and blood pressure as well as reducing the neutral fat of the liver. In addition, the present invention provides an oral composition (food composition, pharmaceutical composition) containing the lactic acid bacterium or a fermented product thereof for the above purpose, that is, reducing the neutral fat of the liver, and reducing the neutral fat of the liver. In addition, it aims to prevent and ameliorate metabolic syndrome from both aspects of lipid metabolism and blood pressure.

  The present invention includes the following aspects.

(I) Lactobacillus brevis derived from Tsuda turnip (I-1) Lactobacillus brevis from Lactobacillus brevis derived from Tsuda turnip having a liver neutral fat reducing action in vivo .
(I-2) In addition to the liver neutral fat reducing action in vivo ,
Γ-aminobutyric acid producing action in the presence of glutamic acid , and
The lactic acid bacterium according to (I-1), which has an action of reducing the total amount of cholesterol in the liver and blood .
(I-3) A lactic acid bacterium (Lactobacillus brevis) derived from Tsuda turnip described in (I-2) having an action of decreasing the amount of LDL cholesterol in blood and increasing the amount of HDL cholesterol. (I- 4 ) The lactic acid bacterium according to (I-2), which has the action of γ-aminobutyric acid based on the action of producing γ-aminobutyric acid (GABA).
(I- 5 ) The action of γ-aminobutyric acid (GABA) is at least one selected from the group consisting of an antihypertensive action, a relaxing action, and a renal function improving action, as described in (I-4) Lactic acid bacteria.
(I- 6) Lactobacillus brevis Lactobacillus from turnip said Tsuda Lactobacillus brevis) is a Lactobacillus brevis 119-2 (L. Brevis 119-2 ), one of the (I-1) ~ (I -5) Lactic acid bacteria described in the above.

(II) Utilization of lactic acid bacteria derived from Tsuda turnip (II-1) Oral composition containing Lactobacillus brevis lactic acid bacterium or culture thereof described in any one of (I-1) to (I- 6 ) (however, Tsuda turnip And food processed foods).
(II-2) (I- 1) to (I- 6) Liver triglyceride reducing agent containing the culture containing viable cells or the live bacterial cells of Lactobacillus brevis lactic acid bacteria according to any of , Liver neutral fat accumulation preventive agent, or cholesterol lowering agent .
(II-3) (I- 1) to (I- 6) to an oral formulation comprising a culture of Lactobacillus brevis lactic bacteria described in any of viable cells of the culture the lactic acid bacteria and γ -An oral preparation for reducing liver neutral fat, suppressing blood pressure increase, and lowering cholesterol, which contains aminobutyric acid .
(II-4) Foods and drinks having a pharmaceutical form selected from the group consisting of tablets, capsules, powders, granules, pills, solutions, suspensions, emulsions, and syrups, particularly supplements (II- Oral preparations described in 3).

  Since the lactic acid bacteria of the present invention have a liver neutral fat reducing action, by continuously ingesting this, the accumulation of neutral fat in the liver caused by ingesting a high cholesterol diet is suppressed, and fatty liver Can be prevented. The lactic acid bacterium of the present invention has an effect of suppressing blood pressure increase based on GABA production action in addition to an effect of reducing liver neutral fat, and an action of lowering blood and liver cholesterol (total cholesterol lowering action, LDL cholesterol lowering action) Therefore, it is possible to reduce risk factors such as arteriosclerosis and hypertension and effectively use them for the prevention of metabolic syndrome.

  Therefore, the lactic acid bacterium of the present invention or a culture thereof suppresses accumulation of neutral fat in the liver, serves as a functional food material for preventing fatty liver, and prevents metabolic syndrome such as arteriosclerosis and hypertension. It is effective as a functional food material.

  In addition, the oral composition of the present invention containing such a lactic acid bacterium or a culture thereof suppresses the accumulation of neutral fat in the liver and serves as a functional food for preventing fatty liver, such as arteriosclerosis and hypertension. It is effective as a functional food for preventing metabolic syndrome.

(1) Lactic acid bacteria administration group (Tsukabu-derived isolated lactic acid bacteria + high cholesterol diet administration group), (2) Control group (high cholesterol diet administration group), and (3) Standard section (basic diet administration group) liver slices Is an image observed under a microscope after staining with hematoxylin and eosin. White spheroids show lipid droplet-like endoplasmic reticulum. Tsuda Turnip from separating lactic acid bacteria (L. Brevis 119-2) of γ- aminobutyric acid (GABA) shows the results indicating the productivity (Experiment 2). (A) shows the HPLC chromatogram which analyzed the amino acid of the supernatant of the culture medium after culture | cultivation, and a comparison control medium (medium before culture | cultivation), respectively. (B) shows the density | concentration (mM) of (gamma) -aminobutyric acid (GABA) and glutamic acid in a culture solution accompanying culture | cultivation. Tsuda Turnip from separating lactic acid bacteria (L. Brevis 119-2) shown cholesterol concentration ([mu] g / ml) in the medium after incubation with cholesterol-containing medium (Experimental Example 3). Cholesterol concentration (μg / ml) in the medium without lactic acid bacteria is used as a control value. Numerical values indicate mean ± standard deviation, and different symbols indicate a significant difference at a significance level of 5%. 2 shows the bile acid deconjugating effect of L. brevis 119-2, L. acidophilus ATCC43121, and L. plantarum NRIC1918 (Experimental Example 4 (1)). (A) shows the chromatogram which measured the residual amount of the added sodium taurocholate (arrow) by using the culture medium after microbial cell removal for HPLC. (B) shows the result of measuring the concentration of cholic acid contained in the culture medium after removal of the cells. In the figure, the control is the result of the culture medium without addition of lactic acid bacteria. In the figure, the numerical value indicates the mean value ± standard deviation, and different symbols indicate that there is a significant difference at a significance level of 5%. As test bacteria, L. Brevis 119-2, L. Plantarum NRIC1918, and L. Using acidophilus ATCC43121, shows the results of examining the cholesterol-lowering effect of the living and dead bacteria bodies (Experiment 4 (2)). In the figure, the numerical value indicates the mean value ± standard deviation, and different symbols indicate that there is a significant difference at a significance level of 5%. With respect to lactic acid bacteria ( L. brevis 119-2), changes over time in the amount of cholesterol in the microbial cells and medium during the culturing process of viable cells are shown (Experimental Example 4 (3)). For Tsuda Turnip from separating lactic acid bacteria (L. Brevis 119-2, and L. Brevis 119-6), were cultured with cholesterol-containing medium, and stained with filipin III, a photograph image obtained by fluorescence observation. (A) an image of the lactic acid bacteria (L. brevis 119-2), (c ) is an enlarged view thereof (× 1000), is an image of (b) is Lactobacillus (L. Brevis 119-6). Lactic acid bacteria showing an (L. Brevis 119-2) and control bacteria (Lactobacillus rhamnosus GG bacteria (ATCC53103)) digestion solution resistance test results performed on. (A) is the result of an artificial gastric juice resistance test, and (B) is the result of an artificial bile powder resistance test (Experimental Example 5 (1)). Results of adhesion tests on intestinal epithelial cells performed on lactic acid bacteria ( L. brevis 119-2), control bacteria ( Lactobacillus rhamnosus GG bacteria (ATCC53103)), and negative control bacteria ( Lactobacillus lactis NBRC12007) 5 (2)). (A) is a microscope observation image showing the attachment of bacteria for intestinal epithelial cells [(a) lactic acid bacteria (L. Brevis 119-2), ( b) L. Rhamnosus GG bacteria (control bacteria), (c) Lc. Lactis NBRC12007 (Negative control bacteria)] and (B) show the number of bacteria attached to intestinal epithelial cells (Experimental Example 2). The number of adherent bacteria is displayed as the number of bacteria per cell culture well area.

1. Lactic acid bacteria of lactic acid bacteria present invention derived from the turnip Tsuda, derived from a plant belonging to a is Tsuda Co., Ltd. (English name / Turnip · Brassica rapa) isolated Lactobacillus from (Lactobacillus) brevis (brevis) species Matsue, Shimane Prefecture of specialty products A lactic acid bacterium, one of which was named Lactobacillus brevis 119-2 ( L. brevis 119-2). Hereinafter, in the present specification, the lactic acid bacteria targeted by the present invention are collectively referred to as “the lactic acid bacteria of the present invention” including the above L. brevis 119-2.

In addition, the lactic acid bacteria of this invention can be isolated from the root of Tsuda turnip (raw) by the following screening method, and have the characteristics described later. Lactic acid bacteria of the present invention, including L. brevis 119-2, are stored in a state that can be sold at the Shimane Prefectural Industrial Technology Center located at 1 Hokuryo, Matsue City, Shimane Prefecture. In addition, “Indication of identification: Lactobacillus brevis 119-2” is issued by the National Institute of Technology and Evaluation (NPMD), an independent administrative agency with an address in 2-5-8, Kazusa Kamashi, Kisarazu, Chiba, Japan. The name has been deposited domestically on January 23, 2013 (deposit date) (accession number: NITE P-1518).

(1) Characteristics of the lactic acid bacteria of the present invention The characteristics of the lactic acid bacteria of the present invention when statically cultured at 37 ° C. for 3 days on an MRS agar medium (pH 6.5) are as follows. .

Based on the results of homology search with 16S rRNA gene using primers with the following sequences, the lactic acid bacteria of the present invention were found to be Lactobacillus in light of the above properties in light of Bergey's Manual of Systematic Bacteriology. It was identified as a strain belonging to brevis .

  In addition to the above mycological characteristics, the lactic acid bacterium of the present invention has a liver neutral fat reducing action as described later (see Experimental Example 1). Furthermore, the lactic acid bacterium of the present invention has a γ-aminobutyric acid producing action (see Experimental Example 2) and a cholesterol-lowering action (reducing action of total cholesterol, LDL cholesterol-lowering action) (Experimental Example) in addition to the liver neutral fat reducing action. 3 and 4). Further, as shown in Experimental Example 5 described later, the lactic acid bacterium of the present invention is resistant to digestive fluid (gastric juice, bile acid) and has adhesion to intestinal epithelial cells. For this reason, when the lactic acid bacterium of the present invention is orally ingested, it reaches the intestine in a living state and stays in the intestine for a certain period of time, which is useful as a probiotic bacterium.

(2) Screening Method for Lactic Acid Bacteria of the Present Invention The lactic acid bacteria of the present invention having a liver neutral fat reducing action can be isolated from the Tsuda turnip by performing the following screening operations (a) to (c).
(A) The root (raw) of Tsuda turnip (English name / Turnip / Brassica rapa) is crushed, and the aqueous extract of the crushed product is cultured on a lactic acid bacteria culture agar medium (MRS) containing calcium carbonate under aerobic conditions. Culture at 72 ° C. for 72 hours, and select white colonies that form halos around them. This is examined by Gram staining and confirmed to be lactic acid bacteria.

(B) Mycological characteristics of the lactic acid bacteria isolated above (bacterial morphology, Grack staining, motility, spores, catalase activity, attitude to oxygen, growth temperature, growth pH, lactic acid fermentation format, optical rotation, sugar utilization The lactic acid bacteria belonging to Lactobacillus brevis having the characteristics described in Table 1 are selected.

(C) Lactic acid bacteria belonging to L. brevis obtained above are orally ingested together with a cholesterol-containing basic feed (high cholesterol diet) by a non-human test animal (for example, a mouse) (test group). For example, after ingesting for 2 weeks or more, the liver is removed, and the following (i) and / or (ii) is compared with the liver of the control group (test animal that ingested a high cholesterol diet without administering lactic acid bacteria) To do.
(I) Number and size of liposecting endoplasmic reticulum in liver (ii) Neutral fat content in liver (mg / g liver)
At least one of (i) and (ii), preferably both, in the lactic acid bacteria belonging to L. brevis subjected to the test, in the liver, with the index being lower in the test group than in the control group A lactic acid bacterium having an action of reducing fat is isolated.

  Furthermore, in addition to the liver neutral fat reducing action, the lactic acid bacterium of the present invention having both GABA productivity and cholesterol lowering action can be obtained by performing the following screening operation in addition to the above (a) to (c). Can be isolated from

  (D) The subject lactic acid bacteria are cultured in an MRS liquid medium containing 5% sodium glutamate. The culture solution is subjected to amino acid analysis, and lactic acid bacteria that produce GABA (GABA-producing lactic acid bacteria) are isolated by the orthophthalaldehyde method or the like using the presence or absence of γ-aminobutyric acid (GABA) in the culture solution as an indicator. .

  (E) A lactic acid bacterium having a cholesterol lowering effect is isolated from the target lactic acid bacterium using the presence or absence of the cholesterol lowering effect as an index. The cholesterol lowering action may be evaluated by an in vitro method (for example, see Experimental Example 3 (1)) in which a target lactic acid bacterium is cultured in a culture medium to which cholesterol is added, and the decrease in cholesterol concentration in the medium is used as an index. In addition, non-human test animals (eg, mice) (test group) orally ingest the subject lactic acid bacteria together with a cholesterol-containing basic feed (high cholesterol diet), for example, after ingesting for 2 weeks or more, blood or / And the amount of cholesterol in the liver (total cholesterol amount) is measured, and it is an indicator that the amount of cholesterol is lower than that of the comparative control group (the test animal ingesting a high cholesterol diet without administering lactic acid bacteria) The in vivo method (for example, see Experimental Example 3 (2)) may be used. The in vivo method using an animal can be employed for further verification of the lactic acid bacteria selected by conducting an in vitro primary screening.

  The order of (c) to (e) is not particularly limited, and (c) → (d) → (e), (d) → (e) → (c), or (e) → (d). → (c), (e) → (c) → (d), or (d) → (c) → (e).

  Details of each operation of the screening steps (a) to (e) will be described in an experimental example described later.

(3) Oral composition of this invention The oral composition of this invention makes it essential requirements to contain the lactic acid bacteria of this invention as an active ingredient. The composition is prepared in the form of a food or drink or a pharmaceutical form using an appropriate edible carrier (food material) in the same manner as a normal food or drink. In addition, the composition is prepared in a pharmaceutical form using an appropriate pharmaceutically acceptable excipient or diluent as in the case of ordinary pharmaceutical products.

  Note that the lactic acid bacterium of the present invention is particularly viable if it has at least a liver neutral fat reducing action, preferably a GABA producing action and a cholesterol lowering action in addition to the liver neutral fat reducing action. It is not necessary to be a bacterium, and it may be sterilized by an ordinary general heat sterilization operation. However, it is known that lactic acid bacteria are effective for maintaining health and longevity by intestinal regulation and intestinal microbiota improvement by being ingested as live bacteria, as is generally known as yogurt. ing. For this reason, it is preferable that the lactic acid bacterium of the present invention is also incorporated into the oral composition of the present invention as a living bacterium.

  The lactic acid bacteria of the present invention can also be blended in the oral composition of the present invention, for example, as a culture of each lactic acid bacterium, a crude or purified product of the culture, or a freeze-dried product thereof. The culture can be typically obtained, for example, by culturing at 37 ° C. for about 48 hours under aerobic conditions using a medium suitable for the lactic acid bacteria of the present invention, such as MRS medium. The cells can be obtained by centrifuging the culture after culturing for about 10 minutes at 3,000 rpm, for example. These can be purified according to conventional methods. Furthermore, the cells can be lyophilized. The lyophilized cells thus obtained can also be used as an active ingredient of the composition of the present invention. In the present specification, these cultures of the lactic acid bacteria of the present invention, crude or purified products of the culture, and freeze-dried products thereof are collectively referred to as “lactic acid bacteria of the present invention and cultures thereof”.

  The oral composition of the present invention can further contain an appropriate amount of nutritional components suitable for maintenance and growth of the lactic acid bacteria of the present invention, if necessary. Specific examples of the nutritional component include carbon sources such as glucose, starch, sucrose, lactose, dextrin, sorbitol, and fructose that are used in the culture medium for culturing each microorganism; nitrogen sources such as yeast extract and peptone Each ingredient such as vitamins, minerals, trace metal elements and other nutritional components can be mentioned. Examples of vitamins include vitamin B, vitamin D, vitamin C, vitamin E, vitamin K, and the like. Examples of trace metal elements include zinc and selenium. Examples of other nutritional components include various oligosaccharides such as dairy oligosaccharide, soybean oligosaccharide, lactulose, lactitol, fructooligosaccharide, and galactooligosaccharide. The amount of these oligosaccharides to be blended is not particularly limited, but usually it is preferably selected from an amount range of about 1 to 3% by weight in the oral composition of the present invention.

Preparation of the oral composition of the present invention into a food and drink form and a pharmaceutical form can follow conventional methods. In addition, the carrier used in the preparation of each of these forms may be any of an edible carrier and a carrier such as a pharmaceutically acceptable excipient or diluent. Details of the pharmaceutical form and pharmaceutically acceptable excipients and diluents available for its preparation are described in detail in the section “Pharmaceutical Form Composition” below. In addition, details of preparation to food and drink forms and edible carriers that can be used at that time will be described in the section of “Food and Drink Form Compositions” below. Particularly in the case of food and drink products, a carrier having a good taste-improving effect is preferable. In general, the blending amount of lactic acid bacteria in the oral composition of the present invention can be appropriately selected from the amount that gives about 10 ⁸ to 10 ¹¹ bacteria in 100 g of the oral composition of the present invention. The number of bacteria is calculated by applying a diluted sample to an agar medium for culturing bacteria, culturing at 37 ° C., and counting the number of grown colonies. Since the number of bacteria and turbidity correlate, if the correlation between the number of bacteria and turbidity (absorbance at a wavelength of 650 to 660 nm) is obtained in advance, the number of bacteria can be counted by measuring the turbidity. it can. The blending amount of the lactic acid bacterium is based on the above amount as a guideline and the form of the oral composition of the present invention to be prepared, and the intended action and effect (liver neutral fat reducing action or liver neutral fat reducing action in addition to GABA It can be appropriately changed depending on the production action [blood pressure increase inhibiting action, etc.] and cholesterol lowering action).

  The oral composition of the present invention preferably contains the lactic acid bacterium of the present invention in a live state. In this case, in formulating the oral composition, it is preferable to avoid adopting severe conditions for lactic acid bacteria such as heating and pressurization. For example, when preparing an oral composition in solid form, it is preferable to directly formulate the lactic acid bacterium of the present invention as a lyophilized cell or process the lyophilized cell with an appropriate coating agent.

(3-1) Pharmaceutical Form Composition The oral composition of the present invention is formed into a general pharmaceutical preparation form using an appropriate pharmaceutical carrier that is pharmaceutically acceptable together with the lactic acid bacteria of the present invention as an active ingredient. Can be prepared. Examples of the pharmaceutical carrier include diluents or excipients such as fillers, extenders, binders, moisturizers, disintegrants, surfactants, lubricants and the like that are usually used in this field. it can. These are appropriately selected and used depending on the dosage unit form of the desired preparation.

  Various dosage forms can be selected as the dosage unit form of the pharmaceutical preparation. Typical examples thereof include solid preparations such as tablets, pills, powders, granules, microcapsules and capsules, and liquid preparations such as liquids, suspensions, syrups and emulsions.

  In the case of forming into a tablet form, examples of the above-mentioned preparation carrier include excipients such as lactose, sucrose, sodium chloride, glucose, urea, starch, calcium carbonate, kaolin, crystalline cellulose, silicic acid, potassium phosphate; water, ethanol, Binders such as propanol, simple syrup, glucose solution, starch solution, gelatin solution, carboxymethylcellulose, hydroxypropylcellulose, methylcellulose, polyvinylpyrrolidone; sodium carboxymethylcellulose, carboxymethylcellulose calcium, low-substituted hydroxypropylcellulose, dried starch, sodium alginate , Disintegrants such as agar powder, laminaran powder, sodium bicarbonate, calcium carbonate; polyoxyethylene sorbitan fatty acid esters, sodium lauryl sulfate, Surfactants such as the monoglycerides of thee; disintegration inhibitors such as sucrose, stearin, cocoa butter, hydrogenated oil; absorption enhancers such as quaternary ammonium bases and sodium lauryl sulfate; humectants such as glycerin and starch; Adsorbents such as lactose, kaolin, bentonite, and colloidal silicic acid; lubricants such as purified talc, stearate, boric acid powder, and polyethylene glycol can be used.

  Furthermore, the tablets can be made into tablets with ordinary coatings, for example, sugar-coated tablets, gelatin-encapsulated tablets, enteric-coated tablets, and film-coated tablets as necessary. A preferable preparation is an enteric tablet or enteric capsule preparation form, whereby lactic acid bacteria can reach the intestine without being invaded by gastric acid.

  In the case of molding into a pill form, as a preparation carrier, for example, excipients such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, kaolin, talc; binders such as gum arabic powder, tragacanth powder, gelatin, ethanol; Disintegrants such as laminaran and agar can be used.

  Furthermore, the oral composition having the preparation form of the present invention may contain a colorant, a preservative, a fragrance, a flavoring agent, a sweetening agent, and the like, if necessary.

The amount of the lactic acid bacterium of the present invention to be blended in the oral form of the pharmaceutical form may be an amount that exhibits the action of the lactic acid bacterium, and can be appropriately selected from a wide range. Usually, it is preferable to contain about 10 ⁷ -10 ¹² in a single dosage unit form of the preparation.

  The administration method of the above preparation is not particularly limited, and is determined according to various preparation forms, the age, sex and other conditions of the subject patient, the degree of disease, and the like.

  The dosage of the above preparation is appropriately selected according to the usage, age of the subject patient, sex and other conditions, the degree of disease, etc. Usually, the amount of the lactic acid bacterium of the present invention which is an active ingredient is approximately about 1 kg of body weight per day. The dosage is preferably about 0.5 to 20 mg, and the preparation can be administered to humans in 1 to 4 divided doses per day. In addition, the preparation of the present invention is preferably ingested (administered) continuously in order to effectively obtain the effect. For example, a mode of taking (administering) continuously or intermittently for 2 weeks or more can be exemplified.

  When an oral composition having the pharmaceutical form of the present invention is ingested (administered), the accumulation of neutral fat in the liver is suppressed based on the liver neutral fat reducing action of the lactic acid bacteria of the present invention in the composition. Is possible. That is, the oral composition of the present invention can be effectively used as a liver neutral fat reducing agent or a liver neutral fat accumulation preventing agent.

  The oral composition having the pharmaceutical form of the present invention is based on the liver neutral fat reducing action, GABA producing action and cholesterol lowering action of the lactic acid bacterium of the present invention in the composition by ingesting (administering) it. Thus, it is possible to suppress the accumulation of neutral fat in the liver, suppress an increase in blood pressure, and decrease or suppress the increase in blood and liver cholesterol levels. That is, the oral composition having the pharmaceutical form of the present invention can be effectively used as a comprehensive preparation for the purpose (efficacy / effect) of reducing liver neutral fat, suppressing blood pressure elevation, and lowering cholesterol.

(3-2) Food / beverage form composition As specific examples of the oral composition of the present invention in the form of a food / beverage product, other than Tsuda turnip itself or processed food of Tsuda turnip (for example, processed foods such as pickled vegetables and sprinkles) If it is a thing, it will not specifically limit. Preferably, preparation forms such as solid preparations (tablets, pills, powders, granules, microcapsules, capsules, etc.) containing the lactic acid bacteria of the present invention, and liquid preparations (solutions, suspensions, syrups, emulsions, etc.) Foods and beverages having gum; sweets such as gum, caramel, gummy, and nougat; fermented foods and beverages such as fermented milk, lactic acid bacteria beverages, fermented vegetable beverages, fermented fruit beverages, and fermented soymilk beverages; and the above fermented foods and beverages such as pudding and bavaroa Examples include dairy products other than products. In the present specification, the terms “fermented milk” and “lactic acid bacteria beverage” conform to the definitions of the former Ministry of Health and Welfare “Ministerial Ordinance on Components of Milk and Dairy Products” Article 37, 37 “Fermented Milk” and 38 “Lactic Acid Beverages”. Shall. That is, “fermented milk” refers to a milk or dairy product made into a paste or liquid obtained by fermentation with lactic acid bacteria or yeast. Accordingly, the fermented milk includes a yogurt form as well as a beverage form. The “lactic acid bacteria beverage” refers to a beverage obtained by diluting milk or a dairy product into a paste or liquid obtained by fermenting with lactic acid bacteria or yeast as a main ingredient.

  Such fermented foods and drinks can be produced according to a conventional method. For example, in a liquid such as a suitable raw material for fermentation containing a nutrient source of lactic acid bacteria, such as vegetables, fruits, and soy milk (soybean emulsion), lactic acid bacteria can be produced. It can be produced by culturing and fermenting the raw material.

Fermentation using lactic acid bacteria is preferably performed by preparing a starter in advance, inoculating the raw material for fermentation and fermenting it. Here, as a starter, for example, a lactic acid bacterium of the present invention is inoculated into a raw material for fermentation that has been sterilized at 90 to 121 ° C. for about 5 to 20 minutes in advance and 10% skimmed milk powder to which a yeast extract has been added. And cultured. The starter thus obtained usually contains about 10 ⁷ -10 ⁹ cells / g culture of the lactic acid bacteria of the present invention.

  The above fermentation raw materials include, as necessary, fermentation promoting substances for good growth of the lactic acid bacteria of the present invention, for example, carbon sources such as glucose, starch, sucrose, lactose, dextrin, sorbitol, fructose, yeast extract, Nitrogen sources such as peptone, vitamins and minerals can be added.

Inoculum of lactic acid bacteria are generally bacterial cells fermentation material containing solution 1mL of about 1 × 10 ⁶ or more is suitable be selected from amounts that preferably is intended to include 1 × 10 ⁷ cells before and after . The culture conditions are generally selected from a fermentation temperature of about 20 to 45 ° C, preferably about 25 to 37 ° C, and a fermentation time of about 5 to 72 hours.

  The lactic acid fermented product obtained as described above may have a card-like form (yoghurt or pudding-like form), and this can be taken as a solid food as it is. The carded lactic acid fermented product can be made into a desired beverage form by further homogenizing it. This homogenization can be carried out using a general emulsifier (homogenizer). By such homogenization, a beverage having a smooth texture can be obtained. In addition, in this homogenization, appropriately diluted as necessary, organic acids for pH adjustment are added, and beverages such as sugars, fruit juices, thickeners, surfactants, and fragrances are produced. Various additives that are usually used can be added as appropriate. The beverage thus obtained can be aseptically filled into a suitable container to obtain a final product.

The amount of intake (administration) is appropriately determined according to the age, sex, body weight, degree of disease, etc. of the living body ingesting it, and is not particularly limited. In general, the amount of lactic acid bacteria is preferably selected from the range of about 10 ⁶ to 10 ⁹ cells / mL. The product can generally be prepared so that about 50 mL to 1,000 mL of it is taken per person per day.

  Other preferred specific examples of the composition of the present invention in the form of food and drink include supplement forms, specifically solid preparations such as tablets, pills, powders, granules, microcapsules and capsules, liquids, suspensions There may be mentioned foods and drinks having the form of liquid preparations such as suspensions, syrups and emulsions.

  In the same manner as the pharmaceutical form composition described above, the preparation is generally used as a preparation carrier, such as a filler, a bulking agent, a binder, a moistening agent, a disintegrant, a surfactant, a lubricant, etc. It is possible to produce using a diluent or excipient. The prescription and manufacturing method according to various forms are basically the same as those of the pharmaceutical form composition, and the above description can be incorporated herein.

The amount of the lactic acid bacterium of the present invention to be blended in the oral composition in the form of food or drink may be an amount that exerts the action of the lactic acid bacterium and can be appropriately selected from a wide range. Usually, it is preferable to contain about 10 ⁷ -10 ¹² in a single dosage unit form of the preparation.

  The administration method of the above preparation is not particularly limited, and is determined according to various preparation forms, the age of the subject, sex and other conditions, the degree of disease, and the like.

  The intake of the above preparation is appropriately selected depending on its usage, subject's age, sex and other conditions, the degree of disease, etc. Usually, the amount of the lactic acid bacterium of the present invention which is an active ingredient is approximately about 1 kg per body weight per day. The dosage is preferably about 0.5-20 mg, and the preparation can be taken in 1 to 4 divided doses per day. Moreover, it is preferable to ingest the formulation of this invention continuously, in order to acquire the effect effectively. For example, the aspect of ingesting continuously or intermittently over 2 weeks can be illustrated.

  By taking (administering) the oral composition having the form of food or drink according to the present invention, accumulation of neutral fat in the liver is achieved based on the liver neutral fat reducing action of the lactic acid bacteria of the present invention in the composition. It is possible to suppress. That is, the oral composition which has the food-drinks form of this invention can be used effectively as a liver triglyceride reducing agent or a liver triglyceride accumulation preventive agent.

  Moreover, the oral composition which has the form of the food / beverage products of this invention, when it is ingested (administration), in the liver triglyceride reduction effect | action of the lactic acid bacteria of this invention in this composition, GABA production effect | action, and cholesterol reduction effect | action Based on this, it is possible to suppress the accumulation of neutral fat in the liver, suppress an increase in blood pressure, and decrease or suppress the increase in blood and liver cholesterol levels. That is, the oral composition which has the food-drinks form of this invention can be effectively used as a comprehensive formulation for the purpose (efficacy / effect) of liver neutral fat reduction, blood pressure rise suppression, and cholesterol reduction.

Thus, the oral composition of the present invention having a form of food or drink includes a health supplement, a health functional food, a food for specified health use, which is used for “reducing liver neutral fat” or “preventing liver neutral fat accumulation”, Or the foods and drinks which have a specific function and are eaten for the purpose of maintaining health, such as supplements. In addition, the oral composition of the present invention having a form of food and drink is used for “suppression of blood pressure increase” and “reduction of cholesterol” in addition to “reduction of neutral liver fat” or “prevention of accumulation of neutral fat in liver”. Food and drink that has a specific function and is eaten for the purpose of maintaining health, such as a health supplement, a health functional food, a food for specified health use, or a supplement.

  Hereinafter, the present invention and its effects will be described in detail in experimental examples. However, the present invention is not limited to the following examples and experimental examples.

Experimental example 1
It was found that lactic acid bacteria having an action of reducing the neutral fat of the liver were present in lactic acid bacteria ( Lactobacillus brevis ) isolated from Tsuda turnip, and the lactic acid bacteria were isolated.

(1) Isolation and identification of lactic acid bacteria ( Lactobacillus brevis ) The root (raw) of Tsuda kabu (English name / Turnip / Brassica rapa) is crushed, and the aqueous extract of the crushed material is used as a plate medium for lactic acid bacteria culture containing calcium carbonate ( MRS). This was cultured under aerobic conditions at 37 ° C. for 48 hours, and white colonies that formed halos were examined by Gram staining and confirmed to be Gram-positive and Neisseria gonorrhoeae. Furthermore, catalase activity (negative), fermentation format (hetero), 15 ° C. growth (+), presence or absence of acid generation from various sugars (fructose fermentation: +, ribose fermentation: +, mannose fermentation: −), and sugar In light of the test results of fertility (see Table 1 above) in light of the Bergey's Manual of Systematic Bacteriology, and from the results of homology searches with 16S rRNA genes, they were isolated from Tsuda kabu The bacterium was identified as a lactic acid bacterium belonging to Lactobacillus brevis .

(2) Confirmation test of liver neutral fat reduction effect Ingestion of the lactic acid bacteria isolated in (1) to the test animal, histopathological examination, and measurement of the amount of neutral fat per gram of liver, A lactic acid bacterium having an action of reducing sex fat was obtained.

(I) Test animal Rat / Jcl: SD / male 4-week-old obtained from CLEA Japan was used as a test animal. Rats that had been bred for one week and that had no abnormalities in the general state were used in this study (11 animals per group). Breeding conditions are: temperature 20-26 ° C, relative humidity 30-80%, lighting time 12 hours (lights on at 8 am, lights off at 8 pm), individually housed in FRP rat bracket cage, feed is mouse / rat during pre-breeding -Feed for hamster (CRF-1; Oriental Yeast Co., Ltd.) was freely ingested. Drinks were given free access to tap water.

(Ii) Preparation of test lactic acid bacteria Lactic acid bacteria ( L. brevis ) obtained in (1) were shake-cultured at 37 ° C using MRS liquid medium at 50 rpm, washed with sterile water, and then lyophilized. (Manufactured by ULVAC) and stored in a desiccator installed in a cool and dark place until the test.

(Iii) Test method and results a) Setting of test animal group Test animals are classified into (1) lactic acid bacteria administration group (lactic acid bacteria + high cholesterol diet administration group), (2) control group (high cholesterol diet administration group), and (3 ) Divided into 3 groups (11 each group) of standard group (basic feed administration group), (1) 5% (dry wt / wt) lactic acid bacteria and cholesterol-containing basic diet (high cholesterol diet) (2) The control group was allowed to freely ingest only the cholesterol-containing basic diet (high cholesterol diet), and (3) the standard group was allowed to ingest only the basic diet for 14 days.

b) Histopathological examination The liver (outer left lobe) was excised from each of the above-mentioned test animals on the 14th day after free intake, and a part thereof was excised and fixed in a 10% neutral buffered formalin solution. The fixed liver was made into a paraffin block and sliced using a microtome. Thin sections were stained with hematoxylin / eosin (H / E staining) and observed with an optical microscope. As a result, it was confirmed that lactic acid bacteria having an action of reducing liver lipid-like endoplasmic reticulum exist. This was obtained as a lactic acid bacterium having a liver fat reducing action and named Lactobacillus brevis 119-2 (L. brevis 119-2). The action of lactic acid bacteria to reduce lipid droplet-like endoplasmic reticulum was judged based on the fact that the number and size of lipid droplet-like endoplasmic reticulum was significantly reduced compared to those in the control group. .

The results of microscopic observation by hematoxylin and eosin staining of liver sections of (1) lactic acid bacteria administration group, (2) control group, and (3) standard group ingesting the lactic acid bacteria ( L. brevis 119-2) As shown in FIG. As can be seen, it was observed that the number and size of lipid droplet-like vesicles clearly decreased in the lactic acid bacteria-administered group compared with the control group.

c) Liver lipid test
(1) The liver (external left lobe) was extracted from test animals in the lactic acid bacteria administration group and (2) control group ingesting the lactic acid bacteria ( L. brevis 119-2), and lipid extraction was performed. For extraction of lipids in the liver, triglyceride E-Test Wako (manufactured by Wako Pure Chemical Industries, Ltd.) (GPD / DAOS method) was used with reference to the method of Folch et al. The amount was calculated. As a result, (1) the amount of neutral fat per gram of liver in the lactic acid bacteria administration group was 50.10 ± 16.04 mg, which was confirmed to be lower than (2) 53.94 ± 17.34 mg in the control group. From these results, it was confirmed from the liver lipid test that lactic acid bacteria ( L. brevis 119-2) have an action of reducing the neutral fat of the liver.

Experimental Example 2 Confirmation of γ-aminobutyric acid (GABA) productivity MRS liquid to which lactic acid bacteria ( L. brevis 119-2) isolated from Tsuda turnip in Experimental Example 1 were added with sodium glutamate to a concentration of 5% The medium (Becton, Dickinson and Company, the same applies hereinafter) was inoculated and cultured at an initial pH of 5.5 at 30 ° C. for 7 days. The obtained culture supernatant (cultured medium) and control medium (precultured medium) were each subjected to amino acid analysis, and NBD-F (7-Fluoro-4-nitrobenzo-2,1,3 The amount of γ-aminobutyric acid (GABA) and glutamic acid in each medium was measured by the -oxadiazole method.

  The results are shown in FIG. (A) shows the HPLC chromatogram of amino acid analysis, and (B) shows the transition of the amount of γ-aminobutyric acid (GABA) and glutamic acid in the culture medium together with the culture time. As can be seen from this result, the lactic acid bacterium (L. brevis 119-2) isolated in Experimental Example 1 consumes glutamic acid in the medium to produce γ-aminobutyric acid (GABA), that is, lactic acid bacterium (L. It was confirmed that brevis 119-2) has a γ-aminobutyric acid (GABA) producing action.

Experimental Example 3 Cholesterol-lowering action (1) Cholesterol-lowering action in culture medium Lactic acid bacteria ( L. brevis 119-2) isolated from Tsuda turnip in Experimental Example 1 were cultured in MRS liquid medium at 37 ° C for 24 hours. After shaking culture at 50 rpm and repeating this three times, the medium was removed, and the 10-fold diluted bacterial solution was adjusted with physiological saline so that the absorbance was OD ₆₅₀ = 0.15. This was inoculated into 0.2% taurocholic acid-containing MRS-THIO supplemented with 10 mg / ml cholesterol ethanol solution to a final concentration of 70 μg / ml to a final concentration of 1% (v / v), and was subjected to anaerobic conditions at 37 ° C. And cultured with shaking at 50 rpm for 24 hours (gas pack system; manufactured by BBL, anero pack; manufactured by Mitsubishi Gas Chemical). After incubation, the mixture was centrifuged at 4400 ° C. for 10 minutes at 5400 rpm, cholesterol contained in the supernatant was extracted with hexane, and then hexane was dried. A 0.2% OPA acetic acid solution and sulfuric acid were added thereto, the absorbance at a wavelength of 550 nm was measured, and the cholesterol concentration in the culture medium was determined based on a calibration curve prepared from standard cholesterol at a known concentration.

The results are shown in FIG. 3 together with the amount of cholesterol in the culture medium in the lactic acid bacteria-free group (control). As can be seen from FIG. 3, it was confirmed that lactic acid bacteria ( L. brevis 119-2) have a significant effect of reducing the amount of cholesterol in the culture medium (significant level is judged to be significant at 5%). .

(2) Cholesterol lowering action in blood and liver (i) Cholesterol lowering action in blood In Experimental Example 1 (2) (iii) c), (1) Lactic acid bacteria ingested L. brevis 119-2 The test animals in the administration group (n = 11) and (2) the control group (n = 11) were fasted for 18 to 23 hours on the 14th day after free intake of each feed, and then sevoflurane (sevoflurane; Maruishi Pharmaceutical Co., Ltd.). ) Under inhalation anesthesia, heparinized sodium blood was obtained from the abdominal aorta and centrifuged (3000 rpm, 10 min, 4 ° C.) to obtain plasma. Using the obtained plasma, total cholesterol (TCHO) (GOD / POD method), HDL cholesterol (HDLC) (dextran sulfate Mg precipitation method / GOD / POD method), and LDL cholesterol (LDLC) (calculated from TCHO and HDLC) ) Was measured.

  The results are shown in the table below.

From this result, it was confirmed that lactic acid bacteria ( L. brevis 119-2) have the effect of reducing not only the neutral fat of the liver but also the total cholesterol.

(Ii) Effect of lowering cholesterol in liver In Experimental Example 1 (2) (iii) c), (1) lactic acid bacteria administration group (n = 11) ingesting lactic acid bacteria ( L. brevis 119-2), and (2 ) The amount of total cholesterol contained in the liver (outer left lobe) extracted from each test animal of the control group (n = 11) was measured. For the measurement of the total cholesterol amount, the total cholesterol amount (mg) per 1 g of the liver was calculated using Cholesterol E-Test Wako (manufactured by Wako Pure Chemical Industries) (cholesterol oxidase / DAOS method). As a result, (1) total cholesterol amount per gram of liver in the lactic acid bacteria administration group was 50.14 ± 12.53 mg (mean value ± standard deviation), and (2) 62.51 ± 12.92 mg (mean value ± standard deviation) of the control group (P <0.05).

From this result, it was confirmed that lactic acid bacteria ( L. brevis 119-2) have the effect of reducing not only the neutral fat of the liver but also the total cholesterol.

Experimental Example 4 Evaluation of cholesterol lowering action
L. Acidophilus, L. Plantrum, L. Cholesterol lowering effect of lactic acid bacteria, such as reuteri has been reported to be due to bile acid deconjugation activity (Non-Patent Document 10 to 12). That is, bile acids having an emulsifying action are decomposed by an enzyme produced by lactic acid bacteria, and cholesterol with reduced solubility is excreted outside the body, thereby lowering the cholesterol level in the body. In addition, Kimoto et al. Reported that Lc. Lactis (live bacteria) excreted cholesterol together with the cells by incorporating cholesterol into the membrane of the cells, and that its activity was significantly reduced in dead cells ( Non-patent document 13).

Therefore, in order to investigate the mechanism of cholesterol lowering effect of lactic acid bacteria ( L. brevis 119-2) isolated in Experimental Example 1, bile acid deconjugation, cholesterol lowering effect of living and dead cells, The amount of cholesterol was quantitatively observed and the surface of the cells was observed under a microscope.

(1) Evaluation of bile acid deconjugation action The bile acid deconjugation action of lactic acid bacteria ( L. brevis 119-2) was evaluated according to the method described in Non-Patent Document 14. In addition to lactic acid bacteria ( L. brevis 119-2), L. acidophilus ATCC43121 and L. plantrum NRIC1918, which are known to have a cholesterol-lowering action based on bile acid deconjugation, were used as test bacteria.

Specifically, the test bacteria were first cultured with shaking in MRS liquid medium at 37 ° C. for 24 hours (50 rpm), and this was repeated three times for stabilization. Next, after removing the medium and washing the cells with sterilized water, the 10-fold diluted bacterial solution was suspended again in sterilized water so that the absorbance was OD ₆₅₀ = 0.15, and MRS-THIO containing 0.2% taurocholic acid (0.2% Sodium 1% (v / v) was added to (Thioglycolate) medium. Subsequently, the cells were cultured at 37 ° C. for 24 hours, and the obtained culture solution was adjusted to pH 7.0 with 1N NaOH, adjusted to a constant volume, and centrifuged (3000 rpm × 10 minutes) to remove the cells. Next, 5N hydrochloric acid was added to the culture medium after removing the cells to adjust to pH 1.0, and then the amount was made constant.

An appropriate amount was collected from this, and 3 times the amount of ethyl acetate was added to recover the dissolved cholic acid. Specifically, the added ethyl acetate was dried at 60 ° C. under a nitrogen gas stream, cholic acid was dissolved with 0.05N NaOH, 16N sulfuric acid and 1% furfural were added, and the mixture was reacted at 65 ° C. for 13 minutes. Acetic acid was added and the absorbance (OD ₆₆₀ ) was measured. The cholic acid concentration was measured from the calibration curve.

  The result of the bile acid deconjugation effect of each lactic acid bacterium is shown in FIG. FIG. 4A shows a chromatogram obtained by subjecting the culture medium after removal of the cells to HPLC and measuring the remaining amount of added sodium taurocholate. FIG. 4B shows the result of measuring the concentration of cholic acid contained in the culture medium after removal of the cells. Incidentally, cholic acid is a decomposition product of sodium taurocholate. In the figure, the control is the result of the culture medium with no test bacteria added.

As shown in (A), unlike L. plantrum NRIC1918 and L. acidophilus ATCC43121 in which a decrease in taurocholic acid was observed, the peak of sodium taurocholate was detected in L. brevis 119-2, as in the control, There was no decrease in taurocholic acid. Corresponding to this result, as shown in (B), cholic acid, which is a decomposition product of taurocholate Na, is hardly detected in L. brevis 119-2, and is not detected in L. plantrum RIC1918 and L. acidophilus ATCC43121. Only uniquely detected. The detected cholic acid was 3.4 μmol / ml in both strains, and almost all of the added sodium taurocholate 3.7 μmol / ml was decomposed into cholic acid.

From these results, the cholesterol lowering effect of lactic acid bacteria (L. Brevis 119-2) is, L. Acidophilus, L. Plantrum , and L. Unlike cholesterol-lowering effect of lactic acid bacteria, such as reuteri, not due to bile acid deconjugation activity It was confirmed.

(2) Evaluation of cholesterol-lowering action of living and dead cells In addition to lactic acid bacteria ( L. brevis 119-2), L. acidophilus ATCC43121 and L. plantrum NRIC1918 are used as test bacteria. The cholesterol-lowering effect of dead cells was evaluated.

Remove the medium from the test bacteria that had been pre-cultured twice in MRS liquid medium, and suspend it in sterile water so that the 10-fold diluted solution has an absorbance (OD ₆₅₀ ) of 0.15, and add this to a new MRS liquid medium. 1% (v / v) was added. This was cultured at 37 ° C. for 24 hours, and then autoclaved at 120 ° C. for 20 minutes. The medium was removed and suspended in the same amount of 0.2% sodium taurocholate-containing MRS-THIO, which was designated as “dead cell group”. In the same manner as above, the precultured test bacteria were suspended in sterilized water and added to MRS-THIO containing 0.2% sodium taurocholate at a rate of 1% (v / v), and this was defined as a “viable cell group”. .

  Both test sections were cultured with shaking (10 rpm) at 37 ° C. for 24 hours under anaerobic conditions. After incubation, centrifuge at 4400C for 10 minutes, 5400 rpm (2800 xg), extract cholesterol in the resulting medium with hexane, dry to hexane, and develop color with 0.2% OPA acetic acid solution and sulfuric acid . Absorbance was measured at 550 nm, and the cholesterol concentration in the culture medium was determined from a standard calibration curve.

  The results are shown in FIG. In the figure, the control is the result of the culture medium without addition of lactic acid bacteria.

As can be seen from FIG. 5, the live cells had a significantly lower cholesterol lowering effect than the control, but the dead cells had no cholesterol lowering effect except for L. plantrum . Although L. plantrum showed a significant difference, its cholesterol-lowering effect was negligible compared to live cells.

From this, it was confirmed that the cholesterol-lowering effect of lactic acid bacteria ( L. brevis 119-2) is an action that is uniquely observed in living organisms.

(3) Time course of cholesterol in culture medium and microscopic observation of bacterial cell surface Regarding lactic acid bacteria ( L. brevis 119-2), the amount of cholesterol in the bacterial cell and culture medium during the cultivation process of live cells was measured.

  Cholesterol in the microbial cells was measured by GC / MS after adding the same amount of distilled water to the microbial cells excluding the medium and extracting with hexane as described above. The detection of cholesterol by GC / MS was performed using GC / MS (GCQ Thermo Fisher scientific) under conditions of mobile phase He 1.2 ml / min and splitless. Cholesterol was quantified using 5αcholestan as an internal standard substance.

  The results are shown in FIG. As can be seen, the cholesterol in the medium decreased from the 6th hour of culture, became 20% or less before the culture in 24 hours of culture, and then stabilized. Cholesterol in cells increased from the 6th hour of culture and tended to increase over time up to 27 hours. Cholesterol was slightly detected in the precipitate collected from the centrifuge tube during centrifugation. Precipitates tended to increase at 24 hours compared to 6 and 9 hours. There was almost no difference over time in the total amount of cholesterol, and cholesterol in the medium tended to migrate to the cells over time.

7A and 7C show photographic images obtained by fluorescence observation of lactic acid bacteria ( L. brevis 119-2) stained with Filipin III after culturing in a cholesterol-containing medium. As can be seen from this, in the lactic acid bacteria (L. brevis 119-2), fluorescence was observed in the cells and it was confirmed that cholesterol was taken into the cells. Moreover, it was observed that the fluorescence was unevenly distributed throughout the cells. On the other hand, even with L. brevis derived from Tsuda turnip, no fluorescence was observed in the cells ( L. brevis 119-6) that did not show cholesterol lowering action (FIG. 7 (b)).

From the above results, the cholesterol-lowering mechanism of lactic acid bacteria (live bacteria) ( L. brevis 119-2) is similar to that of Lc . Lactis (live bacteria) and is due to the incorporation of cholesterol into the cells. It is considered that cholesterol taken into the cells is excreted together with the cells to reduce the amount of cholesterol in the body.

Experimental Example 4 Evaluation of Probiotics Probiotic bacteria are defined as living and reaching the intestines and acting well on the host. Therefore, in order to be a probiotic, lactic acid bacteria taken orally must be resistant to digestive fluid and must stay in the intestine for a certain period.

Therefore, in order to confirm that the lactic acid bacterium ( L. brevis 119-2) isolated in Experimental Example 2 is a probiotic bacterium, a digestive fluid resistance test (see Non-Patent Document 2), and intestinal epithelial cells An adhesion test (see Non-Patent Document 15) was performed.

(1) Digestive fluid resistance test As a digestive fluid resistance test, a resistance test for artificial gastric juice and artificial bile fluid was performed according to the method described in Non-Patent Document 2.

Specifically, first, lactic acid bacteria ( L. brevis 119-2) were cultured in MRS liquid medium at 37 ° C. for 4 hours, and the passage was repeated three times. The artificial gastric juice resistance test was performed by culturing the above lactic acid bacteria ( L. brevis 119-2) at 37 ° C for 4 hours in 0.32% pepsin (110 units / 100 ml) -containing MRS liquid medium adjusted to pH 3.0, 2.5 and 2.0, respectively. Thereafter, the viable cell count was measured using an MRS agar medium. Artificial bile fluid resistance test is performed in an MRS liquid medium containing 0.13, 0.25, 0.5, or 1.0% bile powder (Oxygall Difco) at 37 ° C for 16 hours, and the absorbance (OD ₆₅₀ ) of the cultured medium is measured. It was measured. Artificial gastric juice resistance and artificial bile powder resistance were evaluated using Lactobacillus rhamnosus GG bacteria (ATCC53103) as a control bacteria.

The result of the artificial gastric juice resistance test is shown in FIG. Under pH 2.0 conditions corresponding to fasting gastric juice, both of the control bacteria became 10 ⁴ / ml or less within 1 hour of culture, but at pH 2.5 and 3.0 corresponding to the pH during and after meals, Even after 4 hours of culturing, the number of viable bacteria was about the same as that of the control bacteria, indicating resistance to artificial gastric juice.

The result of the artificial bile powder resistance test is shown in FIG. The growth of control bacteria was inhibited depending on the concentration of bile powder, while the growth of lactic acid bacteria ( L. brevis 119-2) tended to be accelerated depending on the concentration of bile powder. .

(2) Adhesion test to intestinal epithelial cells Adhesion test to intestinal epithelial cells was performed according to the method described in Non-Patent Document 15.

Specifically, intestinal epithelial cells (CaCo-2 cells) were first seeded at a concentration of 1.25 × 10 ⁵ / ml in wells of a 24-well plate and cultured for 15 days after confluence. To this intestinal epithelial cell-containing well, 1 mL of a test bacterium (1.5 × 10 ¹⁰ / ml) previously diluted 100-fold with an antibiotic-free medium is added and cultured at 37 ° C. in a 5% carbon dioxide atmosphere for 1 hour. Attached to cells. In addition to lactic acid bacteria (L. brevis 119-2) and Lactobacillus rhamnosus GG bacteria (ATCC53103) (control bacteria) as test bacteria, Lactobacillus lactis NBRC12007, which has low adhesion to intestinal epithelial cells, is a negative control. Used as a fungus.

  After culturing, the supernatant of the medium was removed, and the cells were washed with PBS. Then, the cells were detached from the test bacteria with a 0.05% Trypsin-EDTA solution, and the number of bacteria was measured on an MRS agar medium. Similarly, intestinal epithelial cells (Caco-2 cells) were cultured using chamber slides (2 well, manufactured by Nalgen), and the test bacteria were attached, followed by fixation with methanol and Gram staining (crystal violet) treatment. After that, it was observed with a microscope.

The microscopic observation image is shown in FIG. 9 (A), and the number of bacteria attached to the intestinal epithelial cells is shown in FIG. 9 (B). In addition, the number of adherent bacteria is displayed as the number of bacteria per cell culture well area. (A), the of (a), (b), and (c) each lactic acid bacteria (L. Brevis 119-2), L . Rhamnosus GG bacteria (control bacteria), and Lc. Lactis NBRC12007 (negative control bacteria) Shows adhesion to intestinal epithelial cells. As shown in (A), adhesion of bacterial cells to the cell surface was observed under the microscope in (a) lactic acid bacteria ( L. brevis 119-2) and (b) L. rhamnosus GG (arrows in the figure). On the other hand, in (c) Lc.lactis NBRC12007 which is a negative control bacterium, adhesion to cells was not observed. (B), the similar to the microscopic observation, Lc. Lactis NBRC12007 number of attached bacteria (negative control bacteria) whereas was only a 10 ² cfu / well, lactic acid bacteria (L. brevis 119-2) is As many as 10 ⁶ cfu / well were detected, similar to L. rhamnosus GG (control).

From this, it was confirmed that lactic acid bacteria ( L. brevis 119-2) have high adhesion to intestinal epithelial cells (Caco-2 cells).

From the results of (1) and (2) above, the isolated lactic acid bacterium of the present invention ( L. brevis 119-2) reaches the intestine in a living state, and adheres to intestinal epithelial cells and stays in the intestinal tract for a certain period of time. The possibility is high, and it is thought that this is useful as a probiotic bacterium.

NITE P-1518

  SEQ ID NOs: 1 and 2 show the primer sequences used in the homology search with the 16S rRNA gene conducted to determine the genus species of the lactic acid bacteria of the present invention.

Claims (6)

Has the effect of taking the cholesterol cells, Tsuda Lactobacillus brevis lactic bacteria L from turnip actobacillus brevis 119-2 (accession number: NITE P-1518) or a culture thereof. Liver neutral fat reducing action in vivo,
Γ- aminobutyric acid production activity in the presence of glutamate, and has the effect of lowering the total cholesterol in the liver and blood, is the culture of lactic acid bacteria or from turnip Tsuda of claim 1. Reducing the LDL cholesterol level in the blood, according to claim 1 or 2 the culture was lactic bacteria from strain Tsuda or described has the effect of increasing the HDL cholesterol level. Claim 1 was lactic bacteria or according to any one of the three oral composition comprising the culture in a state of viable bacteria (except where Tsuda lines and food processing thereof.). Claim was lactic bacteria or described in 1 to 3 either contain the culture in the form of viable bacteria, the oral compositions used in the liver triglyceride reducing or liver neutral fat accumulation prevention applications. Furthermore, the oral composition described in Claim 5 used for the use of a cholesterol fall and a blood pressure rise suppression.

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