JP7316802B2 - Composition for promoting sugar absorption - Google Patents

Description

TECHNICAL FIELD The present invention relates to a composition for enhancing absorption of sugar in the intestinal tract containing lactic acid bacteria and/or bifidobacteria as an active ingredient.

Carbohydrates are digested and absorbed, circulate in the blood, and serve as an energy source for tissues throughout the body. Especially in the brain, since glucose in the blood is the main energy source, an extreme lack of carbohydrates can lead to dizziness, lightheadedness, lack of concentration, and in serious cases, disturbance of consciousness.

Insufficient blood sugar occurs when the amount of sugar intake is constantly insufficient due to, for example, extreme dieting or eating disorders, or when sugar consumption becomes excessive due to exercise on an empty stomach. sell. It has also been reported as a side effect of insulin preparations and oral hypoglycemic drugs used by diabetic patients.

Malabsorption of sugar in the intestinal tract can also cause sugar deficiency. Glucose uptake in the intestinal tract is mainly mediated by monosaccharide transporters expressed in intestinal epithelial cells. Three monosaccharide transporters, the sodium-dependent unidirectional transporter SGLT1 and the facilitative bidirectional transporters GLUT2 and GLUT5, are expressed in the small intestine. contributes to the uptake of sugar in When the expression of these monosaccharide transporters is inhibited, sugar uptake from the intestinal tract is suppressed. For example, it has been reported that tomatoside A, which is a tomato seed-derived saponin, suppresses sugar uptake by suppressing the expression of SGLT1 in intestinal epithelial cells (Patent Document 1). In addition, it has been reported that quercetin contained in apples and onions and apigenin contained in celery and parsley both have the effect of significantly reducing the gene expression of GLUT2 and GLUT5 and suppress sugar uptake (non-patented). Reference 1).

Promoting sugar uptake in the intestinal tract is considered to be one of the means for improving sugar deficiency. However, while many food products and pharmaceutical compositions exhibiting an effect of suppressing sugar uptake have been reported, almost no food products or pharmaceutical compositions that promote sugar uptake have been reported.

By the way, lactic acid bacteria and bifidobacteria are bacteria that have been used in the production of fermented foods since ancient times, and recent studies have revealed that they contribute to the enhancement of various health functions. Lactobacillus cultures have also been suggested to affect sugar absorption. Patent Document 2 discloses that a fermented milk kefir extract (molecular weight of 1000 or less) made using various microorganisms including lactic acid bacteria contributes to the promotion of sugar uptake into myotube cells. However, none of the documents disclose or suggest whether the cells of lactic acid bacteria or bifidobacteria per se are involved in promoting sugar uptake in the intestinal tract.

JP 2017-192312 A Patent No. 3813808

Journal of Functional Foods (2017), 36, 429-439

An object of the present invention is to provide a novel composition for promoting sugar absorption in the intestinal tract.

The present invention provides the following new composition for promoting sugar absorption.
<1> For promoting absorption of sugar in the intestinal tract containing, as an active ingredient, bacterial cells belonging to the genus Lactobacillus, Lactococcus, Leuconostoc or Bifidobacterium Composition.
<2> Bacteria belonging to the genus Lactobacillus, Lactococcus, Leuconostoc or Bifidobacterium are Lactobacillus acidophilus, Lactobacillus amylovorus, Lactobacillus Lactobacillus delbrueckii subsp. bulgaricus , Lactobacillus delbrueckii subsp. lactis, Lactobacillus helveticus, Lactobacillus johnsonii, Lactobacillus paracasei subspecies paracasei (Lactobacillus paracasei subsp. paracasei), Lactobacillus plan Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactococcus lactis subsp. moris), Lactococcus lactis subsp. lactis, Leuconostoc mesenteroides subsp. mesenteroides, Leuconostoc pseudomesenteroides, Bifidobacterium longum longum), Bifidobacterium adolescentis, Bifidobacterium adolescentis Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium catenulatum, Bifidobacterium pseudocatenulatum cterium pseudocatenulatum), Bifidobacterium pseudolongum (Bifidobacterium The composition for promoting sugar absorption in the intestinal tract according to <1>, wherein the composition is one or more selected from (1) selected from (1) pseudolongum).
<3> Bacteria belonging to the genus Lactobacillus, Lactococcus, Leuconostoc or Bifidobacterium are Lactobacillus acidophilus JCM1132, Lactobacillus amylovorus JCM1126, Lactobacillus del brueckii subsp. bulgaricus JCM1002, Lactobacillus delbrueckii subsp. lactis JCM1012, Lactobacillus helveticus JCM1120, Lactobacillus johnsonii JCM2012, Lactobacillus paracasei subsp. paracasei JCM8130, Lactobacillus plantarum JCM1149, Lactobacillus reuteri JCM1112, Lactobacillus rhamnosus JCM1136, Lactococcus lactis subsp. cremoris JCM16167, Lactococcus lactis subsp. lactis JCM5805, Leuconostoc mesenteroides subsp. mesenteroides JCM6124, Leuconostoc pseudomesenteroides JCM9696, Bifidobacterium longum JCM1217, Bifidobacterium adolescentis JCM1275, Bifidobacterium bifi dum JCM1255, Bifidobacterium breve JCM1192, Bifidobacterium catenulatum JCM1194, Bifidobacterium pseudocatenulatum JCM1200, Bifidobacterium pseudolong <2> characterized by being one or more selected from um JCM1205 3. The composition for promoting sugar absorption in the intestinal tract according to .
<4> A feed composition for promoting sugar absorption, a pharmaceutical composition for promoting sugar absorption, or a food composition for promoting sugar absorption, comprising the composition for promoting sugar absorption according to any one of <1> to <3> thing.

According to the present invention, lactic acid bacteria belonging to the genus Lactobacillus, Lactococcus and Leuconostoc and bifidobacteria belonging to the genus Bifidobacterium are used as active ingredients. It is possible to provide a composition for promoting sugar absorption in the intestinal tract.

Gene expression of various glucose transporters (SGLT1, GLUT2, GLUT5) after culturing for 24 hours by adding various heat-killed cells of lactic acid bacteria or bifidobacteria to human colon cancer-derived cell line Caco-2 cultured like intestinal epithelium It is the graph which compared the quantity.

(lactic acid bacteria and bifidobacteria)
The lactic acid bacteria and bifidobacteria of the present invention are lactic acid bacteria classified into the genus Lactobacillus, Lactococcus and Leuconostoc, and bifidobacteria classified into the genus Bifidobacterium. Anything can be used.
Specifically, Lactobacillus acidophilus, Lactobacillus amylovorus, Lactobacillus delbrueckii subsp. bulgaricus ), Lactobacillus delbrueckii subsp. lactis ), Lactobacillus helveticus, Lactobacillus johnsonii, Lactobacillus paracasei subsp. paracasei, Lactobacillus paracasei subsp. Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactococcus lactis subsp. cremoris, Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. Leuconostoc mesenteroides subsp. mesenteroides , Leuconostoc pseudomesenteroides, Bifidobacterium longum, Bifidobacterium adolescentis, Bifidobacterium bifidum (Bifidobacterium adolescentis) idobacterium bifidum), Bifidobacterium breve ( Bifidobacterium breve), Bifidobacterium catenulatum, Bifidobacterium pseudocatenulatum, Bifidobacterium pseudocatenulatum acterium pseudolongum) and the like, but are limited to these isn't it.

The lactic acid bacteria of the present invention include Lactobacillus acidophilus JCM1132, Lactobacillus amylovorus JCM1126, Lactobacillus delbrueckii subsp. bulgaricus JCM1002, Lactobacillus delbrueckii subsp. lactis JCM1012, Lactobacillus helveticus JCM1120, Lactobacillus johnsonii JCM2012, Lactobacillus paracasei subsp. paracasei JCM8130, Lactobacillus plantarum JCM1149, Lactobacillus reuteri JCM1112, Lactobacillus rhamnosus JCM1136, Lactococcus lactis subsp. cremoris JCM16167, Lactococcus lactis subsp. lactis JCM5805, Leuconostoc mesenteroides subsp. mesenteroides JCM6124, Leuconostoc pseudomesenteroides JCM9696, Bifidobacterium longum JCM1217, Bifidobacterium adolescentis JCM1275, Bifidobacterium bifi dum JCM1255, Bifidobacterium breve JCM1192, Bifidobacterium catenulatum JCM1194, Bifidobacterium pseudocatenulatum JCM1200, Bifidobacterium pseudolong um JCM1205 is preferred.
The above strains can be obtained from RIKEN BioResource Center (Tsukuba, Ibaraki, Japan) and the like.

(Preparation of lactic acid bacteria and bifidobacteria)
Lactic acid bacteria and bifidobacteria may be cultured according to a conventional method for culturing each bacterium, and a desired amount thereof may be prepared. An example of preparation is shown below. MRS medium (Difco) was used for lactic acid bacteria belonging to the genus Lactobacillus, M17 medium (Difco) was used for lactic acid bacteria belonging to the genus Lactococcus, and lactic acid bacteria belonging to the genus Leuconostoc and bifidobacteria belonging to the genus Bifidobacterium were and 1% glucose-containing GAM bouillon (Nissui), and the resulting cultures are collected by centrifugation to obtain cells. The obtained cells may be used as they are, or cells subjected to concentration, drying, or freeze-drying may be used. Cells killed by heat drying or the like can also be used.

(How to Use)
As described above, the composition of the present invention can contain, as an active ingredient, bacterial cells subjected to concentration, drying, freeze-drying, or killed cells obtained by heat drying, etc. It can be widely used as Bacterial cells may be cultured using a formulation, food, drink, or feed as a medium, and the culture medium may be used as it is as a formulation, food, drink, or feed, or bacterial cells cultured in another medium may be used as a formulation, food, drink, or feed. Or you may add to feed.

The administration subject of the composition of the present invention is not particularly limited, and can be administered to humans, but the administration subject can also be animals other than humans (eg, dogs, cats, horses, rabbits, etc.). When the subject of administration is a human, it can be administered to a minor under the age of 20, an adult, or an elderly person aged 65 or over.
The intake of the composition of the present invention is individually determined in consideration of the symptoms, age, etc. of the subject of administration, but for adults, it is sufficient to take about 1-5000 mg per day.

(Method for evaluating sugar absorption promoting ability)
It can be evaluated by the method described in Examples.

EXAMPLES The present invention will be described in more detail below based on examples, but the present invention should not be construed as being limited to these examples.

(Example product 1) Heat-killed cells of lactic acid bacteria and bifidobacteria The test bacteria in the following (1) were placed on MRS medium (Difco) for Lactobacillus, M17 medium (Difco) for Lactococcus, and Leuconostoc and Bifidobacterium was inoculated into 1% glucose-containing GAM bouillon (Nissui) and statically cultured at 37° C. for 16 hours. The culture was washed twice with physiological saline and once with sterilized water to obtain washed cells. The washed cells were freeze-dried to obtain a powder of cells. The bacterial cell powder was diluted with sterilized PBS(−) to 10 mg/ml and heated at 80° C. for 30 minutes to obtain heat-killed bacterial cells. The heat-killed cells were diluted to 100 μg/ml with DMEM (Sigma) containing 10% FBS (Gibco), 1× Non-essential Amino Acids (Gibco) and 1% penicillin-streptomycin (Sigma).

(1) Test Bacteria The following 21 strains of the genus Lactobacillus, Lactococcus, Leuconostoc, and Bifidobacterium were used as test bacteria.
Lactobacillus acidophilus JCM1132, Lactobacillus amylovorus JCM1126, Lactobacillus delbrueckii subsp. bulgaricus JCM1002, Lactobacillus delbrueckii subsp. lactis JCM1012, Lactobacillus helveticus JCM1120, Lactobacillus johnsonii JCM2012, Lactobacillus paracasei subsp. paracasei JCM8130, Lactobacillus plantarum JCM1149, Lactobacillus reuteri JCM1112, Lactobacillus rhamnosus JCM1136, Lactococcus lactis subsp. cremoris JCM16167, Lactococcus lactis subsp. lactis JCM5805, Leuconostoc mesenteroides subsp. mesenteroides JCM6124, Leuconostoc pseudomesenteroides JCM9696, Bifidobacterium longum JCM1217, Bifidobacterium adolescentis JCM1275, Bifidobacterium bifi dum JCM1255, Bifidobacterium breve JCM1192, Bifidobacterium catenulatum JCM1194, Bifidobacterium pseudocatenulatum JCM1200, Bifidobacterium pseudolong um JCM1205
The above strains can be obtained from RIKEN BioResource Center (Tsukuba, Ibaraki, Japan) and the like.

[Test Example 1] Test on Effect of Promoting Glucose Transporter Expression by Lactic Acid Bacteria and Bifidobacterium Example Product 1 was subjected to the following test.
Human colon cancer-derived cell line Caco-2 was seeded at 5×10 ⁴ cells/well on a 24-well plate (BD) coated with collagen with Cell matrix type IC (Nitta Gelatin) and added with 10% FBS (Gibco). , 1× Non-essential Amino Acids (Gibco), DMEM (Sigma) containing 1% penicillin-streptomycin (Sigma). The cells were cultured for 24 days while exchanging the medium every 2-3 days to differentiate into an intestinal epithelium. After 24 days, the cells were washed once with PBS, and cultured for 24 hours by adding a medium in which various cells were suspended at 100 μg/ml to the test group and a medium containing no cells to the control group. After 24 hours, the cells were washed once with cold PBS(-), 300 μl of TRIzol Reagent (Invitrogen) was added to the washed cells, and the cells were lysed by pipetting. The cell lysate was collected in a 1.5 ml tube, incubated at room temperature for 5 minutes or longer, and stored at -80°C until RNA extraction.

The cell lysate stored at −80° C. was returned to room temperature and 60 μl (1/5 volume) of chloroform was added. After vigorously shaking and stirring by hand, the mixture was allowed to stand at room temperature for 3 minutes. After centrifugation at 12,000×g and 4° C. for 15 minutes, the upper layer (aqueous layer) was transferred to another tube. An amount of isopropanol equal to that of the aqueous layer was added, mixed by pipetting, and allowed to stand at room temperature for 10 minutes. Centrifuge at 20,000×g for 10 minutes at 4° C. and remove the supernatant, leaving a pellet. 300 μl of 75% ethanol was added, the pellet was washed by inversion mixing, and centrifuged at 20,000×g at 4° C. for 5 minutes. The supernatant was completely removed leaving the pellet and air dried for 30 minutes. The pellet was suspended in 30 μl of Nuclease-Free Water and incubated at 55° C. for 10 minutes to dissolve RNA. The dissolved RNA solution was ice-cooled, and the nucleic acid concentration was measured using BioSpec-nano (Shimadzu Corporation).

cDNA synthesis from RNA was performed according to a scaled-down protocol of ReverTraAce (Toyobo). The total amount of RNA subjected to reverse transcription was 250 ng/μl. The synthesized cDNA was stored at -20°C until quantitative PCR (qPCR).

KAPA SYBR FAST Master Mix (2x) Universal (Kapa Biosystems) was used as a qPCR reaction reagent. StepOnePlus (Applied Biosystems) was used as a qPCR apparatus. The qPCR reaction was performed according to a scaled down version of the KAPA SYBR FAST Master Mix (2x) Universal "ABI Instrument Protocol (Fast Program)". 1 μl/well of 20-fold diluted cDNA was used as a template. The qPCR program used was a partially modified Fast program of the StepOnePlus system (reaction volume: 20 µl/well → 5 µl/well, reaction time for Denature Step: 3 seconds → 5 seconds, reaction temperature for Annealing Step: all 60 °C).

The relative expression levels of SGLT1, GLUT2 and GLUT5 genes were calculated by the ΔΔCT method. The relative expression level of each gene was corrected with the relative expression level of GAPDH, a housekeeping gene. The nucleotide sequences of the primers used in the test are shown below.

(Test results)
FIG. 1 shows the gene expression levels of glucose transporters when Caco-2 cells differentiated into intestinal epithelium-like cells were cultured for 24 hours with the addition of either lactic acid bacteria or 21 strains of bifidobacteria. For SGLT1 and GLUT5, all test strains increased gene expression (SGLT1 about 1.5-2.5 fold, GLUT5 about 1.2-2.7 fold compared to the control group). . As for GLUT2, Lactobacillus amylovorus, Lactobacillus paracasei subspecies paracasei, Lactobacillus reuteri, and Lactobacillus rhamnosus had almost no effect on gene expression, but the other 17 strains did not affect gene expression. increased (approximately 1.2-2.3 fold compared to the control group). Therefore, it was suggested that lactic acid bacteria and bifidobacteria increase gene expression of monosaccharide transporters and increase sugar uptake in the intestinal tract, regardless of bacterial species.

According to the present invention, sugar in the intestinal tract containing as an active ingredient the cells of bacteria belonging to the genus Lactobacillus, Lactococcus, Leuconostoc or Bifidobacterium Absorption-enhancing compositions can be provided.

Claims (3)

A composition for promoting sugar absorption in the intestinal tract containing, as an active ingredient, the cells of a bacterium belonging to the genus Lactobacillus, wherein the bacterium belonging to the genus Lactobacillus is Lactobacillus delbrueckii subspecies bulgaricus. bulgaricus), Lactobacillus delbrueckii subsp.lactis, Lactobacillus helveticus, Lactobacillus johnsonii , Lactobacillus plantarum The composition, which is one or more of Bacteria belonging to the genus Lactobacillus are Lactobacillus delbrueckii subsp. bulgaricus JCM1002, Lactobacillus delbrueckii subsp. Lactis JCM1012, Lactobacillus helveticus us ) JCM1120, Lactobacillus johnsonii JCM2012 , Lactobacillus plantarum JCM1149 The composition for promoting sugar absorption in the intestinal tract according to claim 1, characterized in that it is one or more selected from thing. A feed composition for promoting sugar absorption, a pharmaceutical composition for promoting sugar absorption, or a food composition for promoting sugar absorption, comprising the composition for promoting sugar absorption according to claim 1 or 2.