HK1213010B - Bacterium belonging to genus lactobacillus - Google Patents

Description

Lactobacillus bacteria

Technical Field

The present invention relates to a bacterium belonging to the genus Lactobacillus. More particularly, it relates to a novel lactobacillus pentosus (lactobacillus pentosus).

Background

Lactic acid bacteria and bifidobacteria include substances having excellent physiological activities such as gastrointestinal regulation effect and immunopotentiation effect, and are used for various applications depending on the characteristics of the bacteria. Among them, many reports have been reported in recent years on the progress of research on the development of a weight-reducing effect by taking these bacterial strains into the body.

For example, patent document 1 reports that Lactobacillus rhamnosus ATCC53103 decomposes a lipid (triacylglycerol) causing obesity and inhibits its absorption in the body, and that after living l.brevis KB290, which is one of plant lactic acid bacteria, reaches the intestine, it is known that the intestinal survival rate and intestinal tract reaching ability are excellent (but the number of excreted bacteria is smaller than the number of ingested bacteria) (see non-patent document 1). further, non-patent document 2 reports Lactobacillus acidophilus L-92 that the intestinal tract reaching ability of the strain is excellent when the strain is recovered from feces depending on 93% of the number of ingested bacteria, and non-patent document 3 reports that the intestinal tract survival ability of l.gasseri SBT2055 is investigated and that when 100g of Lactobacillus acidophilus SBT2055 is administered⁶～5×10⁶cfu/g fermented milk, 1 × 10 was detected at most from feces⁵L.gasseri SBT2055 around cfu/g.

On the other hand, the following are reported for bifidobacteria: bifidobacterium animalis subsp. lactis GCL2505 strain has an intestinal reach property that viable bacteria reach the intestinal tract after oral ingestion, and exhibits a significant intestinal proliferation property (see patent document 2). Non-patent document 4 reports as follows: DN-173010, which is about 20% of the number of bacteria taken in feces, was detected when B.animalis ssp.lactis DN-173010 was administered to an adult.

Documents of the prior art

Patent document

Japanese patent application laid-open No. 2011-206057 of patent document 1

Japanese patent application laid-open No. 2011-

Non-patent document

Non-patent document 1 "health function and application of lactic acid bacterium" (Japanese original name "health care function and application of lactic acid bacterium at bone site と") (page 160 and 162 published by CMC (applied date, 8 and 31) at 2007, 8 and 31

Non-patent document 2 journal of the Japanese society for lactic acid bacteria (Japanese name: Japanese society for lactic acid bacteria ) No. 12 2001 "isolation and Properties of Lactobacillus acidophilus L-92 Strain having excellent persistence in human intestinal tracts" (Japanese name: ヒト Ostrin for property of い (better) (とそ) of で strain having residual ability in < 3526 > Oseltia

Nonpatent document 3 Microbiol. Immunol.2006 No. 50 "Monitoring and simulation of Lactobacillus gasseri SBT2055in the human endogenous extract," 867-

Non-patent document 4J. mol. Microbiol. Biotechnol.2008 No. 14 "simulation of Bipolar analysis DN-173010in the mechanical microbial amplification in solubilized form or in a transferred product-a transferred polypeptide in synthesized synthesis of metals additives", page 128-136

Disclosure of Invention

In the past, the intestinal canal accessibility of various probiotics has been studied as described above, but with regard to lactic acid bacteria, the number of bacteria that have been reported to reach the intestinal canal by the number of bacteria equal to or less than the number of ingested bacteria has been limited, or the number of bacteria that reach the intestinal canal has not been measured.

In addition, since fat absorption is mainly performed in the small intestine, bifidobacterium, which generally grows in the large intestine, shows an intestinal proliferation property and thus does not sufficiently suppress fat absorption. Further, although lactic acid bacteria are known to act on the upper end of the large intestine, no bacterial species showing proliferation in the intestine have been reported.

The present invention addresses the problem of providing a lactic acid bacterium that exhibits intraintestinal proliferation.

The present invention relates to Lactobacillus pentosus strain TUA4337L with a deposit number of NITE BP-1479, which is characterized by having an intestinal proliferation ability.

The lactic acid bacterium of the present invention has an excellent effect of proliferating in the intestinal tract. Further, when the lactic acid bacterium of the present invention is ingested, the lactic acid bacterium reaches the intestine and proliferates to maintain the fat absorption inhibitory effect and the like, and the physiological activity of the bacterial cells is enhanced, and further, an excellent effect of obtaining a high weight-reducing effect is obtained.

Drawings

FIG. 1 is a graph showing the results of screening in artificial intestinal juice.

Fig. 2 is a graph showing the progress of weight gain. The "+" marks in the figure indicate significant differences (p <0.05) relative to the high fat diet group.

FIG. 3 is a graph showing the amount of triglycerides in serum. The "+" marks in the figure indicate significant differences (p <0.05) relative to the high fat diet group.

Detailed Description

The lactic acid bacterium of the present invention is lactobacillus pentosus strain TUA4337L, which is characterized by having a growth ability in the intestinal tract. In the present specification, "having a growth ability in an intestinal tract" or "growing in an intestinal tract" means that the cells grow in the small intestine and/or the large intestine, preferably in the small intestine after reaching the intestinal tract, and the degree of growth ability can be evaluated as "having a growth ability" in the following cases: that is, the culture is cultured in artificial intestinal juice at 37 deg.C for 6hr, relative to OD at inoculation₆₆₀Showing a value of 10 times or more.

The present inventors have conducted studies on the proliferation ability of about 480 kinds of lactic acid bacteria retained by the present inventors in artificial intestinal juice, and have found that when a suspension of lactobacillus pentosus selected from them is administered to an animal, the number of excreted bacteria of lactobacillus pentosus strain TUA4337L is significantly increased compared to the number of administered bacteria, and have completed the present invention.

Lactobacillus pentosus strain TUA4337L identified as NRIC 0883 under accession number NITE BP-1479, deposited at the national institute of advanced technology for evaluation of the products (NITE) patent microorganism depositary (Kazusa falciparum 2-5-8, Kyowa prefecture, Kyowa, Japan) on 12/10/2012 of International depositary. Hereinafter, the strain Lactobacillus pentosus TUA4337L will be referred to as TUA4337L strain.

The mycological properties of the TUA43 4337L strain are shown in tables 1 and 2. The assimilation of sugars in Table 2 is determined using the bacterial identification kit API 50CH (BIOMETRIEUX). Furthermore, "+" in Table 2 means assimilable saccharide, and "-" means non-assimilable saccharide.

[ Table 1]

Form of the bacterium	Bacillus
		Gram staining property	Positive for
Movement property	Is free of
		Spore	Is free of
Spores	Is free of
		Catalase reaction	Is free of
Propagation at 15 ℃	○
		Propagation at 40 ℃	○
Aerobic propagation	○
		Anaerobic propagation	○
Propagation pH	3.0～12.5

[ Table 2]

The TUA43 4337L strain is described in detail in examples below, and is characterized by having an increased number of excreted bacteria relative to the number of ingested bacteria, i.e., having an intestinal proliferation activity. The in-intestine growth ability is, for example, a number of bacteria preferably 10 times or more, more preferably 15 times or more, further preferably 20 times or more, and still further preferably 25 times or more, based on the number of bacteria at the start of culture, after culturing in an artificial intestinal juice at 37 ℃ for 6 hours.

In addition, the recA gene sequence (SEQ ID NO: 1) encoded by the DNA extracted from TUA4337L strain was 99% identical to the recA gene sequence of Lactobacillus pentosus IG1 strain. In The present specification, The similarity is expressed by a score using, for example, a search program BLAST which uses an algorithm developed by Altschul et al (The Journal of Molecular Biology, 215, 403-.

The medium for culturing the TUA4337L strain is not particularly limited, and may be a medium containing a carbon source, a nitrogen source, inorganic salts, organic nutrients, and the like. In addition, the culture can be carried out in an agar medium or a liquid medium. The culture temperature is preferably 10 to 45 ℃, more preferably 15 to 42 ℃, further preferably 28 to 38 ℃, further preferably 35 to 37 ℃, and the pH value for proliferation is preferably 3.0 to 12.5, more preferably 3.5 to 12.0.

The strain of the present invention may be in various forms such as a lactic acid bacterium (live bacterium or dead bacterium) itself, a lactic acid bacterium-containing material, and a lactic acid bacterium-treated material. The viable bacteria can be obtained from lactobacillus-containing material such as lactobacillus culture solution. The dead bacteria can be obtained by, for example, heating, ultraviolet irradiation, formalin treatment, acid treatment, or the like of the live bacteria. The obtained viable bacteria and dead bacteria can be further ground and crushed to obtain treated product. In addition, from the viewpoint of sufficiently exerting the growth effect in the intestinal tract, live bacteria are preferable as the lactic acid bacteria in the above-described forms, but dead bacteria may be mixed.

Examples of the lactic acid bacteria include live bacteria, wet bacteria, and dry bacteria. Examples of the lactic acid bacteria-containing material include a lactic acid bacteria suspension, a lactic acid bacteria culture (including a bacterial cell, a culture supernatant, and a culture medium component), and a lactic acid bacteria culture solution (a material obtained by removing a solid component from a bacterial cell culture). Examples of the lactic acid bacteria-treated product include ground products, crushed products, liquid products (such as an extract), concentrates, slurries, dried products (such as a spray-dried product, a freeze-dried product, a vacuum-dried product, and a centrifugally-dried product), and diluted products.

Examples

The present invention will be specifically described with reference to the following examples, but the present invention is not limited to the following examples.

Example 1< screening Using growth Capacity in Artificial intestinal juice as an index >

The present inventors evaluated the growth ability in artificial intestinal juice of about 480 strains (including JCM strain) of lactic acid bacteria mainly comprising a plant-derived lactic acid bacteria among the lactic acid bacteria retained.

Specifically, glycerol-preserved bacteria of each lactic acid bacterium were inoculated at 1 v/v% into MRS medium (Difco laboratories) (10mL), and cultured at 35 ℃ for 16 to 17 hr. Next, the OD of each culture solution was measured with a spectrophotometer UV-1600 (Shimadzu Co., Ltd.)₆₆₀(absorbance at 660 nm), 100. mu.L of MRS medium was used as OD of each culture solution₆₆₀The culture solution prepared for 10 was inoculated into an artificial intestinal solution (10mL) having the composition shown below. Thereafter, the cells were cultured at 37 ℃ for 6hr with slow shaking, and then OD was measured₆₆₀And determining proliferation rate (OD after 6 hr)₆₆₀OD at inoculation₆₆₀). Representative screening results are shown in table 3 and fig. 1.

< Artificial intestinal juice (pH6.45) >

MRS Medium 9mL

1mL of 10 w/v% bile (Wako pure chemical industries, Ltd.) solution

100 μ L of 1 w/v% pancreatin (from Porcine: SIGMA)

In addition, as the bile solution and the pancreatin solution, sterile bile solution and pancreatin solution treated with 0.22 μm filter paper (PVDF membrane, manufactured by Millipore Co., Ltd.) were used.

[ Table 3]

From the results, it was found that lactobacillus pentosus and lactobacillus plantarum tend to increase in proliferation rate, and among them, lactobacillus pentosus strain TUA4337L has particularly high proliferation rate and excellent proliferation performance in intestinal tracts.

Example 2< evaluation of intestinal tube growth ability in vivo >

Specifically, about 10 million cells (1.0 × 10 am) were administered to mice freely ingesting high-fat food using the administration samples prepared as follows (TUA 4337L strain prepared as follows)⁹cells) aliquots (equivalent to 250 μ L of bacterial suspension) of lactic acid bacteria were administered 1 time (n-5) to C57BL/6J mice (10 weeks old, male).

< preparation of administration sample (viable bacteria-containing sample) >

[ 1] Glycerol-storing bacteria of TUA4337L Strain were inoculated at 1 v/v% into MRS medium (30mL)

[ 2] culture (35 ℃, 20hr)

[ 3] the culture was centrifuged (8000rpm, 5min) to remove the supernatant, and the supernatant was suspended in 30mL of PBS (-)

[ 4] the suspension of [ 3] was centrifuged (8000rpm, 5min) and the supernatant was removed, followed by resuspension in 5mL of PBS (-)

[ 5] counting of bacteria by microscope

[ 6 ] A bacterial suspension (prepared from PBS (-) for liquid feeds) was prepared by dividing a 200-hundred million cells solution into 15-mL centrifuge tubes, centrifuging (8000rpm, 5min) to remove the supernatant, and suspending the supernatant in 5-mL liquid feeds (60 kcal% FAT for high FAT Diet: Research Diet)

Thereafter, 2 days of whole-bezoar were collected 4 times (afternoon, and afternoon on the day of the start of the experiment), the number of bacteria in each whole-bezoar was quantified by the following method, and the intestinal increase rate of TUA4337L strain (number of bacteria in each whole-bezoar/number of administered bacteria) in each mouse was calculated. The results are shown in Table 4.

< determination of the number of bacteria by real-time PCR >

[ 1] after adding 1mL of PBS (-) to 100mg of (wet-heavy) bezoar, the mixture was broken by a spatula

[ 2] after collecting 100mg of bezoar using Eppendorf tube (registered trademark), centrifugation (15000rpm, 5min) and removal of supernatant, suspension with 1mL of PBS (-) was performed (operation from centrifugation to suspension was repeated 2 times)

[ 3] after removing the supernatant from the suspension of [ 2], DNA was extracted with a Kit (QIAamp DNA pool Mini Kit: QIAGEN)

(cell disruption was performed by repeating 3 times the procedure of adding 300mg of Glass BEADS (150 to 212. mu.m: SIGMA), 300. mu.L of phenol/chloroform/isoamylalcohol (25: 24: 1) and 900. mu.L of buffer ASL (reagent in kit), centrifuging at 3000rpm for 1min with Multi-BEAS SHOCKER MB-200(YASUI KIKAI), and then standing on ice for 1min)

[ 4] quantification of lactic acid bacteria in intestinal tract contents by real-time PCR under the conditions shown below

(real-time PCR conditions)

(1) 10. mu.L of SYBR Premix Ex Taq ll (Takara Bio), 0.8. mu.L of reach primer (10. mu.M), 0.4. mu.L of ROX reference DyeII, 6. mu.L of sterilized water, and 2. mu.L of the DNA solution were mixed to prepare a PCR reaction solution. The following primers (the 16S rDNA sequences of Lactobacillus pentosus and Lactobacillus plantarum are 100% identical) were used to specifically detect the 16S rDNA of Lactobacillus pentosus and Lactobacillus plantarum.

primer 1: 5'-GCAAGTCGAACGAACTCTGGTATT-3' (Serial number 2)

primer 2: 5'-CGGACCATGCGGTCCAA-3' (SEQ ID NO. 3)

(2) PCR was carried out using 7500Real Time PCR System (Applied Biosystems), 95 ℃ for 30 seconds, 60 ℃ for 5 seconds, and 34 seconds as 1 cycle, and 60 cycles were counted to obtain the number of copies of the content per 1g of the intestinal tract from the fluorescence intensity, the total amount of the content in the intestinal tract, and the dilution ratio.

(3) The copy number of 16S rDNA per 1 cell was also determined and converted into the number of bacteria. In addition, it was confirmed that neither lactobacillus pentosus nor lactobacillus plantarum was detected by the real-time PCR in mice to which no lactobacillus was administered.

[ Table 4]

From the results, it was found that the number of bacteria in excreta of lactobacillus pentosus strain TUA4337L was larger than the number of bacteria taken. From the above, it was demonstrated that Lactobacillus pentosus 4337L strain was passed through the stomach in a viable state, proliferated in the intestinal tract, and excreted.

Example 3< weight gain inhibitory Effect >

C57BL/6J mice (8 weeks old, male) were divided into the following 4 groups: generally, a food group, a high-fat food + live group, a high-fat food + dead group (each n ═ 10), and the foods shown in table 5 below were fed for 32 days, respectively, and the body weights were measured and the average values were calculated every day. The transition of the average value is shown in fig. 2. In addition, group comparisons were performed using a t-test at a significance level of 0.05.

Specifically, each solid food was freely ingested by each group in table 5. The high-fat food + live bacteria group was a sample for administration prepared in the same manner as in example 2, and the high-fat food + dead bacteria group was a sample for administration prepared as follows, and each of the samples was administered with an amount of about 10 hundred million cells per day of lactic acid bacteria. On the other hand, 250. mu.L of PBS (-) without lactic acid bacteria was administered to the diet group, and 250. mu.L of liquid feed without lactic acid bacteria was administered to the high-fat diet group.

[ Table 5]

*10kcal％FAT(Research Diet)

60kcal％FAT(Research Diet)

< preparation of administration sample (sample containing dead bacteria) >

[ 1] Glycerol-storing bacteria of TUA4337L Strain were inoculated at 1 v/v% into MRS medium (30mL)

[ 2] culture (35 ℃, 20hr)

[ 3] the culture was centrifuged (8000rpm, 5min) to remove the supernatant, and the supernatant was suspended in 30mL of PBS (-)

[ 4] the suspension of [ 3] was centrifuged (8000rpm, 5min) and the supernatant was removed, followed by resuspension in 5mL of PBS (-)

[ 5] counting of bacteria by microscope

[ 6 ] A200-hundred million cells solution was dispensed into a 15-mL centrifuge tube, centrifuged (8000rpm, 5min) to remove the supernatant, added with 5mL artificial gastric juice (125mM NaCl, 7mM KCl, pH1.0), stirred, and allowed to stand for 60min

[ 7 ] the solution of [ 6 ] was centrifuged (8000rpm, 5min) to remove the supernatant, and then suspended in 5mL of liquid feed (60 kcal% FAT) to prepare a bacterial suspension

As a result, a significant weight gain inhibitory effect of the TUA4337L live bacteria-administered group was shown relative to the control (high-fat food group). And live bacteria administration is more effective than dead bacteria administration. It is considered that lactobacillus pentosus strain TUA4337L effectively affects the host by proliferating in the intestinal tract.

Example 4< fat absorption inhibitory Effect >

The same contents of food as in example 3 were fed for 2 weeks with the group structure of normal food group, high fat food group, and high fat food + live bacteria group (each n is 12) in example 3. Thereafter, after fasting overnight, olive oil (nacalaitesque) administration (5mL/kg) was performed, and after 3 hours, dissection and serum was collected from the great vein. Triglyceride (TG) in serum was measured by using triglyceride E-test Wako (Wako pure chemical industries, Ltd.). The results are shown in FIG. 3. In addition, group comparisons were judged by t-test for significant differences from a significant level of 0.05.

As a result, a significant tendency to increase TG in blood was confirmed in the high-fat diet group relative to the normal diet group. Therefore, it is considered that if a high-fat food is continuously consumed, it becomes a body which easily absorbs fat. In addition, the inhibition of increase in TG in blood was confirmed in the TUA43 4337L live cell administered group relative to the control (high fat diet group). Therefore, it is presumed that fat absorption inhibition is one of the mechanisms of the weight gain inhibition effect, and that the effect is already effective 1 day after the start of live bacterial administration of TUA4337L, and that the effect is continuously exhibited.

Industrial applicability

Since the lactic acid bacteria of the present invention have a proliferation ability in the intestine, when ingested into the body, they can proliferate after reaching the intestine to continuously suppress fat absorption and effectively suppress weight gain, and thus can be used for the purpose of reducing weight.

Sequence Listing free content

Sequence No. 1 of the sequence listing is the base sequence of recA of Lactobacillus pentosus TUA 4337L.

The sequence number 2 in the sequence table is a base sequence of the lactobacillus pentosus/lactobacillus plantarum specific primer.

Sequence number 3 in the sequence table is a base sequence of the lactobacillus pentosus/lactobacillus plantarum specific primer.

Claims (1)

1. Lactobacillus pentosus strain TUA43 4337L with a deposit number of NITE BP-1479, characterized by having a proliferation potency in the intestinal tract.