KR20080096383A - Novel lactic acid bacteria and various processed products produced by using the lactic acid bacteria - Google Patents

Abstract

A noble lactobacillus is provided to improve probiotics effect by reaching bulk content of microbial to the field when taking the lactobacillus, to increase an extinction ability by generating no gastric acid because the lactobacillus has an excellent acid-resistant, to suppress pathogens as the probiotics and to be used for fermented product, health food, functional food and drug. A noble lactobacillus is characterized in that a) lactobacillus is alive in a buffer solution of pH 1.0 at least for 60 minutes, b) a number of the lactobacillus increases in a short time, c) the lactobacillus suppresses a pathogen d) the lactobacillus promotes a breeding of useful bacteria. The lactobacillus belonging to the Lactobacillus species is Lactobaci11us johnsonii No. 1088.

Description

NOVEL LACTIC ACID BACTERIA AND VARIOUS PROCESSED PRODUCTS PRODUCED BY USING THE LACTIC ACID BACTERIA}

The present invention is a lactic acid bacterium belonging to the genus Lactobaci11us, which has a strong acid resistance that survives at least 60 minutes in a buffer of pH 1.0, proliferates in a short time, inhibits pathogens, and promotes the growth of useful bacteria. It relates to a novel lactic acid bacterium characterized by having a property such as a thing, and a variety of products characterized by processing using the new lactic acid bacteria. In addition, the indication of the percentage in this specification is based on a weight unless there is particular notice.

About the acid resistance of the lactic acid bacteria belonging to the genus Lactobacillus, the test in pH 3.0 is reported with Lactobacillus acidophilus (refer nonpatent literature 1). Moreover, lactic acid bacteria belonging to the genus Lactobacillus are classified into detailed species such as Lactobacillus asidophilus, Lactobaci11us johnsonii, and Lactobacillus gasseri by Fujisawa et al. See Patent Document 2).

On the other hand, the effects of lactic acid bacteria as probiotics have recently attracted attention for the prevention or treatment of allergic inflammatory bowel disease, infectious diseases, ulcerative colitis, hypertension, cholesterol, colon cancer and the like (see Non-Patent Document 3). However, when a conventionally known lactic acid bacterium is used for food and drink, medicine, etc., it is known that the number of bacteria which reach the intestine by the influence of gastric acid at the time of ingestion or intake decreases (refer nonpatent literature 4). Conventionally, in order to prevent the reduction of the number of bacteria, a method of reducing the killing of lactic acid bacteria by gastric acid has been adopted by a method such as coating a lactic acid bacteria powder or a lactic acid bacteria tablet to be used (see Patent Documents 1 to 5). ). In addition, yogurt using Lactobacillus johnsonii having acid resistance is also commercially available.

[Patent Document 1] Japanese Patent Laid-Open No. 5-186335

[Patent Document 2] Japanese Patent Laid-Open No. 5-186336

[Patent Document 3] Japanese Patent Laid-Open No. 5-186337

[Patent Document 4] Japanese Patent Laid-Open No. 2000-16320

[Patent Document 5] Japanese Patent Laid-Open No. 2001-64189

[Non-Patent Document 1] Prasad, J. et al., International Dairy Journal, Vol. 8, pp. 993, 1998

[Non-Patent Document 2] Fujisawa, T. et al., International Journal of Systematic Bacteriology, Vol. 42, No. 3, p. 487, 1992

[Non-Patent Document 3] Gupta, P. A. et al., International Journal of Pour Microbiology (Internationa1 Journa1 of Food Microbiology), Vol. 29, pp. 105, 1996

[Non-Patent Document 4] Hood S. Hood, S. K. et al., Journal of Food Science, Vol. 53, p. 1514, 1988

In view of the above prior art, the present inventors have a strong interest in acid resistant lactic acid bacteria which can reach the intestine without being killed by gastric acid for many years, and as a result of the study of acid resistant lactic acid bacteria, the present inventors have not been able to survive at least for 60 minutes at pH 1.0. The present invention has been completed by finding a novel lactic acid bacterium belonging to the genus Lactobacillus which has excellent acid resistance, and furthermore, finding that it can be used for various products as the use of this novel lactic acid bacterium.

A first object of the present invention is to provide a novel lactic acid bacterium belonging to the genus Lactobacillus which proliferates in a short time, has excellent acid resistance and suppresses the growth of pathogens, and a second object of the present invention is the use of the novel lactic acid bacteria. As an example, to provide a variety of products containing the lactic acid bacteria.

The 1st invention (henceforth a 1st invention) of this invention which solves the said subject is a lactic acid bacterium which belongs to the genus Lactobacillus, and has the following a) -d):

a) having strong acid resistance which survives for at least 60 minutes in a buffer at pH 1.0,

b) multiply in a short time,

c) inhibiting pathogens, and

d) promoting the growth of useful bacteria

It is a novel lactic acid bacterium characterized by having the property of Lactobacillus, and Lactobacillus belonging to the genus Lactobacillus john sony No. It is a preferable aspect that it is 1088 (Accession Number: NITE P-278).

The 2nd invention (henceforth 2nd invention) of this invention which solves the said subject is a lactic acid bacterium which belongs to the genus Lactobacillus, and has the following a) -d):

a) having strong acid resistance which survives for at least 60 minutes in a buffer at pH 1.0,

b) multiply in a short time,

c) inhibiting pathogens, and

d) promoting the growth of useful bacteria

Lactobacillus belonging to the genus Lactobacillus, Lactobacillus Johnsoni No. It is a preferable aspect that it is 1088 (accession number: NITE P-278), and a process is fermentation by a novel lactic acid bacterium, mixing of a new lactic acid bacterium, sequencing of a new lactic acid bacterium, or refining a new lactic acid bacterium.

Next, the present invention will be described in detail. The first invention is a lactic acid bacterium Lactobacillus johnsoni No. 1, which the present inventors isolated from human gastric juice as shown in Test Example 1 described later. 1088 (hereinafter abbreviated as No. 1088), a novel lactic acid bacterium belonging to the genus Lactobacillus, which is not known in the prior art, and can be used for various products. These strains are apparently new strains from the base sequence of 16SrRNA, as is apparent from the test examples described later. In addition, as is apparent from the test examples described later, the cells proliferate in a short time, inhibit the growth of pathogens, promote the growth of useful bacteria, and at least 60 minutes in a buffer solution of pH 1.0 (0.1 M hydrochloric acid / sodium citrate buffer). It is a lactic acid bacterium with excellent acid resistance to survive.

2nd invention is various products containing the novel lactic acid bacteria which belong to the genus Lactobacillus which proliferates in a short time, suppresses the propagation of a pathogen, and has excellent acid resistance of the said 1st invention, Such as yoghurt fermented by the said novel lactic acid bacteria It can be used for many products such as fermented products, health foods made of the freeze-dried powder of the new lactic acid bacteria, and pharmaceuticals such as tablets processed from the new lactic acid bacteria powder.

In order to manufacture the said fermentation product, it carries out as follows, for example. Lactobaci11 bulgaricus and Streptococcus thermophilus are added to the raw material oil as the novel lactic acid bacteria and the starting materials, and can be prepared by a conventional method.

In order to manufacture the said novel lactic acid bacteria powder, it carries out as follows, for example. The novel lactic acid bacteria were inoculated in a commercially available medium (for example, MRS broth, manufactured by Beckton Dickinson) (for example, inoculated at a rate of 1 × 10 ⁷ per 1 ml of medium), and cultured (for example, For example, 12 to 18 hours at 37 ° C., the cells are collected by centrifugation after completion of the culture, and the collected cells are washed with a washing solution (for example, sterile saline solution with a salt concentration of 0.85%), and again. The microbial cells were collected by centrifugation, washed with a solution containing mainly skim milk powder (for example, 10% skim milk powder and 1% sodium glutamate solution), suspended and lyophilized by a conventional method. To prepare.

In order to manufacture the tablet which processed the said novel lactic acid bacteria powder, it carries out as follows, for example. The novel lactic acid bacteria powder is processed into tablets by a conventional method using an excipient consisting of sugar, dextrin, starch and the like, for example. As described above, the novel lactic acid bacteria can be treated or used in the same manner as known lactic acid bacteria.

Next, test examples are given and the present invention will be described in detail.

Test Example 1

This test was performed to isolate and identify novel lactic acid strains belonging to the genus Lactobacillus. In addition, isolates and identification of strains are supervised by the Ministry of Health, Labor and Welfare, "Food Hygiene Testing Guidelines: Microbial Edition", Japan Food Hygiene Association, 1990 and Lactobacillus Research Meeting, "Science and Technology of Lactobacillus", Society Publication Center, It carried out according to the method described in 1996.

1. Isolation of Lactic Acid Bacteria

1) Collection of Strains

Gastric juice was collected from 10 healthy adults, and 9 ml of sterile saline was added to 1 ml of the collected gastric juice to prepare a sample solution. The prepared sample solution was diluted 10 times to 100,000 times with sterile physiological saline to prepare a diluted solution.

0.1 ml of gastric juice solution, sample solution and each diluted solution were applied to the surface of the medium of the chalet containing sterilized biel (BL) agar medium (manufactured by Nissui Pharmaceutical Co., Ltd.) and MRS (MRS) agar medium (Becton Dickinson Company). Smear it using a cone rod, and put each chalet into an anaerobic jar (Mitsubishi Gas Kagakusha, aneropack square ruler) with an anero pack (made by Mitsubishi Gas Kagakusha). It was incubated for 48 hours at 37 ℃. After the completion of the culture, the colonies shown were passaged three generations with MRS agar medium (Becton Dickinson) and pure strains were isolated.

2) Identification of Strains

Identification of the isolated strains was carried out in the Lactobacillus genus according to the method described in the literature, because the gram staining was positive, the cell type was bacillus, the catalase was negative, the motility was negative, the apoptosis was negative, and the lactic acid production was positive. It confirmed that it was a strain belonging, and acquired 15 weeks.

3) Isolation of Acid-resistant Strains

The 15 strains were separately inoculated in MRS broth (manufactured by Beckton Dickinson) and incubated at 37 ° C for 48 hours. 0.1 ml of the obtained bacterial liquid was added to 2 ml of 0.1 M sodium citrate buffer solution at pH 2.0, and maintained at 37 ° C. for 2 hours. The remaining bacterial count was measured, and one strain having the most residual bacterial count was obtained.

2. Identification of Strains

Identification of the obtained strains is described in Lactobacillus Research Conference, "Science and Technology of Lactic Acid Bacteria", Society Publication Center, 1996 and Peter H .. a. Peter H.A. Sneath, "Bergeys Manual of Systematic Bacteriology", Vol. 2, William & Wilkins, 1986.

3. Test result

TABLE 1

 A. Morphological Properties (morphology when grown in MRS agar medium)   1. Shape of the cell  Bacillus   2. Cell size  0.4 ~ 0.5 × 2.0 ~ 4.0㎛   3. Polymorphism of Cells  none   4. Mobility  none   5. Apo Formation  none  B. Cultural Properties     1. Morphology of colonies when grown on MRS agar medium        (1) shape  Round, rough structure        (2) size  2 ~ 3mm        (3) color tone  White        (4) pigment production  none     2. litmus milk  Acid, coagulation  C. Physiological Properties     1. Gram dyeability  positivity     2. Catalase  voice     3. Growth temperature  30 ~ 40 ℃     4. Attitude to Oxygen  Facultative anaerobic

TABLE 2

     5. Production of Acids from Sugars  Positive galactose, glucose, fructose, mannose, mannitol, N-acetylglucosamine, esclin, salicylic acid, cellulose, lactose, saccharose, trehalose, raffinose, tagatose negative glycerol, erythritol, arabinose , Ribose, xylose, adonitol, sorbose, arbutin, myriose, inulin, mellitose, amidone, glycogen, xylitol, fucose, arabitol, tranose, lyxose, arabil Gluconate     6. Degradation Products from Sugars  DL lactic acid 7. Generation of NH ₃ from Arginine  voice     8. Sodium Chloride Resistance  Not grown in the presence of 1% (w / v) NaCl     9. pH tolerance  pH 9.5 Not grown    10. Methylene Blue  Not grown in 0.1% added oil

As a result of this test, the bacteriological properties of the obtained strains are as shown in Table 1 and Table 2. In addition, Table 2 is the continuation of Table 1. As is apparent from Table 1 and Table 2, from bacteriological properties, it was proved that the strain was Lactobacillus zononyi, and Lactobacillus zononyi no. It was named 1088. The strain was deposited on November 14, 2006 to the Patent Microorganisms Depository Center for Product Evaluation Technology Infrastructure, an independent administrative corporation, and was given accession number of NITE P-278.

Test Example 2

This test is no. This was done to investigate the characteristics at the base sequence level of 1088. In addition, No. The identification of the nucleotide sequence of 1088 was made to Technosuruga Co., Ltd., and it was reported that it was identified by the following method.

1. Preparation of test cells

No. 1088 was anaerobicly cultured at 37 ° C for 24 hours using MRS Agar medium (manufactured by Oxoid Co., Ltd.) to prepare test cells.

2. Determination of Sequence

The operation from extraction of base to cycle sequence was performed based on each protocol. Base extraction is performed using an Insta Gene Matrix (manufactured by Biorad Co., Ltd.), and polymerase chain reaction using Prime Star HS DNA Polymerase (manufactured by Takara Bio Co.). For the cycle sequence, a Big Dye Terminatar V3.1 Cycle Sequencing Kit (manufactured by Applied Biosystems) was used. The primers used were 9F, 339F, 785F, 1099F, 536R, 802R, 1242R and 1510R, and the sequence was measured using an ABI PRISV 3100 Genetic Analyzer System (manufactured by Applied Biosystems). Auto Assembler (manufactured by Applied Biosystems) is used for the analysis software, and Apollon DB's latest reference stock database (manufactured by Technosurugas Corp.) and international base sequence database (Gin Bank / DVJ / EMM) is used for homology search. Biel (Gen Bank / DDBJ / EMBL) was used.

3. Test result

TABLE 3

Comparative strain Homology search Homology (%) Lactobacillus johnsonii ATCC 3323 Apollon DB Reference Database (Techno Suruga Shizuoka)  1482/1487 (99.7) Lactobacillus johnsonii NCC 533 International Sequence Database (GenBank / DDBJ / EMBL)  1564/1564 (100.0)

The results of this test are as shown in Table 3 and Sequence Listing. As is apparent from Table 3 No. 1088 has a 99.7% homology with the ATCC33200 stock of Lactobacillus Johnsoni in the Apollon Divi's latest reference stock database (manufactured by Technosuruga Co., Ltd.). It was. Therefore, no. It was confirmed that 1088 was Lactobacillus john soni.

Since all the bacteria have a common nucleotide sequence in the 16Sr nucleotide sequence, the taxonomic group of the bacteria can be estimated by the 16Sr nucleotide sequence. However, in order to estimate the bacterial group belonging to lactic acid bacteria, not only the 16Sr base sequence but also the morphology, physiological properties, and biochemical properties of bacteria are important, as shown in the test examples of the present invention.

Test Example 3

This test is no. It carried out to investigate acid resistance of 1088.

1. Preparation of Sample

No. 1088, Lactobacillus gasteria TI1012 known as acid resistance (derived from human feces. Department of Biodefense Preservation, Department of Medicine, Tokai University), Lactobacillus john sony JCM2012 (obtained from R & D Center), commercially available Lactobacillus Johnsoni LC1, Lactobacillus asidophilus JCM1132 (obtained from the Institute of Science and Technology) and Lactobacillus gasery JCM1131 (obtained from the Institute of Science and Technology), separately from HRS broth (Becton Dickinson) Inoculated) and incubated at 37 ° C for 18 hours to prepare a culture solution of the sample.

In addition, Lactobacillus Johnsoni LC1 was isolate | separated from the commercial yogurt (made by Nestle Corporation) containing this strain as follows. The yoghurt is diluted 10 times, 10 times ³ times, 10 times ⁵ times, and 10 times ⁷ times with sterile saline solution, 0.1 ml of each dilution is added dropwise to a Biel (BL) agar plate medium (manufactured by Niss Co., Ltd.) The mixture was smeared, and anaerobic culture was carried out at 37 ° C. for 2 days. After incubation, each colony was discriminated by a conventional method.

2. Test method

0.1 ml of the six cultures obtained were separately added to 0.1 M hydrochloric acid and citric acid buffer solution of pH 2.0, pH 1.5, and pH 1.0, maintained at 37 ° C, and after 15 minutes, after 30 minutes, after 60 minutes and 90 minutes Thereafter, the number of remaining bacteria was measured by a conventional method over time.

3. Test result

TABLE 4

The test results are shown in Table 4. In addition, ND of Table 4 shows that residual microbe number was not detected. As is apparent from Table 4, No. 1088 showed the highest number of remaining bacteria, and survived even when kept at pH 1.0 for 90 minutes, and was found to be a strain having excellent acid resistance.

Test Example 4

This test is no. 1088 was performed to demonstrate that the strain was different from the Lactobacilus johnsonii LC1 used in Test Example 3 above.

1. Preparation of Sample

1) Preparation of strain

The strains used in Test Example 3 were each inoculated into MRS broth (manufactured by Beckton Dickinson), incubated at 37 ° C for 18 hours, and the respective strains were collected from the culture solution, followed by phosphate buffered saline solution. It wash | cleaned once and prepared each strain.

2) Preparation of deoxyribonucleic acid (DNA)

Each strain was suspended in phosphate buffered saline, respectively, and DNA was extracted from each suspension by Wizard Genomic DNA Purification Kit (promega). The extraction method also followed the method attached to the kit.

3) primer sequence used

TABLE 5

Six primers shown in Table 5 were used.

2. Test method

TABLE 6

TABLE 7

Using the reaction solution shown in Table 6, the reaction cycle was carried out under the conditions shown in Table 7, and electrophoresis was carried out by a conventional method using 1.4% agarose gel with 5 µl of the reaction solution after completion of the reaction.

3. Test result

The result of this test is as showing in FIG. In the figure, M, A and B are 100b rata marker, LC1 strain and No. 1088 strain. As is apparent from FIG. 1, a clear difference was observed in the electrophoretic patterns of P-07, P-09 and P-30, and LC1 strain and No. The 1088 strains all belonged to Lactobacillus john soni, but proved to be different strains.

Test Example 5

This test is no. This was done to investigate the inhibitory effect of 1088 on Helicobacter pylori.

1. Preparation of Sample

Test strain No. JCM2012 (obtained from the Institute of Chemical Science and Technology), the reference stock of 1088 and Lactobacillus Johnsoni, was developed by Helicobacter Pylori No. To a liquid medium containing 5% horse serum added to 100 ml of BHI broth (manufactured by Becton Dickinson) along with 130 (isolated from chronic human gastritis patients. Two strains of the genus Lactobacillus per 1 ml were inoculated at a ratio of 1.0 × 10 ⁶ and Helicobacter pylori at a ratio of 1.0 × 10 ⁷ , and cultured under 37 ° C. aerobic culture conditions, and 6 hours, 12 hours after the start of the culture, The medium was collected over time at 24 hours and 48 hours to prepare a sample. In addition, the liquid medium itself without adding the above bacteria was used as a control sample.

2. Test method

The collected sample was put into aneropack helico (manufactured by Mitsubishi Gas Kagakusha) in an aneropak square ruler (manufactured by Mitsubishi Gas Kagakusha) with the following medium, and cultured in aerobic culture at 37 ° C for 4 days, and then Helicobacter pylori The bacterial count of was measured. The medium used for bacterial count was 7 ml of horse serum, 25 mg of tetrazolium (manufactured by Sigma), 2500 units of polymyxin B (manufactured by Sigma), vancomycin (jig) in 1000 ml of BHI agar medium (manufactured by Beckton Dickinson). 10 mg of masa), 5 mg of bacitracin (manufactured by Sigma), and 2 mg of amphotericin ratio (manufactured by Sigma) were added.

3. Test result

The results of this test are as shown in FIG. In the drawings, reference numeral ● denotes a control sample and Δ denotes a no. 1088, □ designates the reference strain of Lactobacillus john sony. As is apparent from Fig. 2, No. In the 1088-added sample, the bacteria count of Helicobacter pylori after 24 hours was 1.0 × 10 ³ / ml and below the detection limit after 48 hours. On the other hand, in the sample added with Lactobacillus john sony, the number of bacteria of Helicobacter pylori after 24 hours was below the detection limit after 1.0 × 10 ^4.2 / ml and 48 hours. From this test result, No. It has been confirmed that 1088 inhibits Helicobacter pylori bacteria more than 10 times than the reference strain of known Lactobacillus john sony.

Test Example 6

This test was performed using No. No. for enterohemorrhagic E. coli, Escherichia coli O-157. It carried out to investigate the inhibitory effect of 1088.

1. Preparation of Sample

A sample and a control sample were prepared in the same manner as in Test Example 5, except that a liquid medium containing 0.7% glucose added to rubber (GAM) bouillon (manufactured by Nissei Seiyakusha) was used. In addition, Escherichia coli O-157 is a bioisopreservation strain of the Faculty of Medicine, Tokai University, as a clinical isolate.

2. Test method

The bacterial counts of the sample and the control sample were measured by the same method as Test Example 5, except that DHL agar medium (made by Nisui Seiyakusha) was used for culturing Escherichia coli O-157.

3. Test result

The results of this test are as shown in FIG. 3. In the figure, 은 is a control sample, △ is a no. 1088, □ designates the reference strain of Lactobacillus john sony. As is apparent from Fig. 3, No. In the 1088-added sample, the bacterial count of Helicobacter pylori after 24 hours was 1.0 × 10 ^{3 · 8} / ml and below the detection limit after 48 hours. On the other hand, in the sample added with Lactobacillus john sony, the number of bacteria of Helicobacter pylori after 24 hours was below the detection limit after 1.0 × 10 ^4.9 / ml and 48 hours. From this test result, No. It has been confirmed that 1088 inhibits Escherichia coli O-157 at least 10-fold higher than the reference strain of known Lactobacillus johnsonii.

Test Example 7

This test is no. It was performed to investigate the inhibitory effect on Salmonella bacteria of 1088.

1. Preparation of Sample

A sample and a control sample were prepared in the same manner as in Test Example 5. In addition, Salmonella thyphimurium LT2, a clinical isolate, is a bioprotective strain of the Tokai University School of Medicine.

2. Test method

The bacterial count of the sample and a control sample was measured by the method similar to the said Test Example 5.

3. Test result

The results of this test are as shown in FIG. 4. In the figure, 은 represents a control sample and Δ represents a no. 1088, □ represent the baseline of Lactobacillus john sony. As is apparent from Fig. 4, No. In the 1088-added sample, it was below the detection limit of Salmonella after 12 hours. On the other hand, in the reference strain addition sample of Lactobacillus john sony, the number of bacteria of Salmonella after 12 hours was 1.0 × 10 ³ / ml. From this test result, No. It has been confirmed that 1088 inhibits Salmonella more than 1000 times more than the reference strain of known Lactobacillus john sony.

Test Example 8

This test is no. This was done to investigate the inhibitory effect on 1088 Clostridium difficile.

1. Preparation of Sample

A sample and a control sample were prepared in the same manner as in Test Example 5 except that anaerobic culture was performed. In addition, Clostridium difficile JCM1296 was obtained from the Institute of Chemical Research.

2. Test method

The bacterial counts of the sample and the control sample were measured in the same manner as in Test Example 5, except that CCFA agar medium was used for the culture of the Clostridium difficile.

3. Test result

The results of this test are as shown in FIG. In the figure, 은 represents a control sample and Δ represents a no. 1088, □ represent the baseline of Lactobacillus john sony. As is apparent from Fig. 5, No. In the 1088-added sample, Clostridium difficile JCM1296 was detected 1.0 × 10 ³ / ml after 48 hours. On the other hand, in the sample added with Lactobacillus john sony, the bacterial count of Clostridium difficile JCM1296 after 48 hours was 1.0 × 10 ^3.6 / ml. From this test result, No. It has been confirmed that 1088 inhibits Clostridium difficile JCM1296 over the reference strain of known Lactobacillus john sony.

Test Example 9

This test is no. 1088 Candida albicans (Candida a1bicans) to investigate the effect of inhibiting the growth was carried out.

1. Preparation of Sample

A sample and a control sample were prepared in the same manner as in Test Example 5 except that anaerobic culture was performed. Candida albicans TI3001 is a biodiversity preservation strain of the Faculty of Medicine, Tokai University.

2. Test method

Candida a1bicans (Candida a1bicans) The same method as in Test Example 4, except that the number of bacteria inoculation for measuring the number of bacteria of the 300300 was 1.0 × 10 ⁶ / ml and the medium for measuring the number of bacteria was changed as follows The number of bacteria of the sample and the control sample was measured by. As a medium for bacterial count measurement, after sterilizing a potato dextrose agar medium (manufactured by Nissei Seiyakusha), 1.4 ml of 10% tartaric acid was added per 1 liter of medium, and the pH was adjusted to 3.5.

3. Test result

The results of this test are as shown in FIG. In the figure, 은 represents a control sample and Δ represents a no. 1088, □ designates the reference strain of Lactobacillus john sony. As is apparent from Fig. 6, No. In the 1088-added sample, the bacterial count of Candida albicans TI3001 was detected 2 × 10 ⁵ / ml after 48 hours. On the other hand, in the sample of the reference strain addition of Lactobacillus john sony, the number of cells of Candida albicans TI3001 was 5 × 10 ⁵ / ml after 48 hours. From this test result, No. It was confirmed that 1088 suppresses Candida albicans TI3001 rather than the known Lactobacillus john sony.

Test Example 10

This test is no. It was performed to investigate the inhibitory effect on 1088 Helicobacter pylori infected mice.

1. Preparation of Sample

Helicobacter pylori No. 3 cultured in the same manner as in Test Example 4 in 15 sterile mice (Tokai University Department of Medicine biodefense breeding mice). 130 (clinical isolates. Bioprotective preservation strains of the Tokai University Department of Medicine) were orally administered 1.0 × 10 ⁹ /0.5 ml for 3 days, and were bred by a conventional method. After 4 weeks of breeding, 5 mice were slaughtered and Helicobacter pylori no. The 130 number of bacteria was measured by the method described in the following test method, which is also the mouse 1O ⁴ of ⁵ ~1O Helicobacter pylori No. gastric tissue per 1g 130 infection was confirmed, and Helicobacter pylori no. 130 infected mice were made.

2. Test method

Helicobacter pylori No. Ten mice infected with 130 were prepared by the same method as in Test Example 4. 1088 1.0 × 10 ⁹ was orally administered once, and was bred by a conventional method. No. Five infected mice were slaughtered after 1088 administration, two weeks and four weeks, respectively, and the Helicobacter pylori No. The number of bacteria of 130 was measured by the following method. In addition, No. Ten mice which did not receive 1088 were used as control mouse samples.

Helicobacter pylori bacteria in the stomach tissue was measured by anesthetizing mice with ether, removing the stomach by removing the stomach contents, adding sterilized phosphate buffered saline, and glass homogenizer (manufactured by ASAHI TECHNO GLASS CORPORATION). 5 ml volume), and the sample liquid was prepared. This sample solution 1O ^{-1, -2} and diluted with 1O 1O ^-3, and 0.1ml of each dilution is plated on the medium used in Test Example 4, cultured at 37 ℃ US No. 4 to 5, and the number of emerged colonies The number of bacteria was measured by multiplying by the dilution ratio. In addition, this test was performed twice.

3. Test result

The result of this test is as showing in FIG. In the drawings, the black marks are no. The mean value of the control group not administered 1088, the white mark is No. The mean value of the group which administered 1.088 is shown, and the result of the significant difference test (T test) of both groups is shown by p <0.05. As is apparent from Fig. 7, In the control group not receiving 1088, both trials had 1.0 × 10 ^{4 · 7} / g He1icobacter pylori No. 130 existed, but No. In the sample group administered 1088, Helicobacter pylori No. It was confirmed that 130 was 1.0 × 10 ^2.4 / g (first time) and 1.0 × 10 ³ / g (second time), which was significantly lower than the control group. Therefore, No. 1088 Oral Administration of He1icobacter pylori No. It was proved to be effective at inhibiting 130.

Test Example 11

This test is no. This was done to investigate the effect on the intestinal flora of 1088.

1. Preparation of Sample

Ten sterile mice which were the same as in Test Example 10 were orally administered with a diluted solution of 100-fold dilution of a human adult, and bred for 4 weeks by a conventional method to prepare human flora mice.

2. Test method

In 10 human flora mice, No. 1088 was cultured by MRS broth (Becton Dickinson Co., Ltd.) for 24 hours at 37 ° C. (2.0 × 10 ⁹ / ml of bacteria) orally administered once 0.5 ml daily, and bred by a conventional method. Four weeks later, the bacterial flora in the feces of each mouse was detected by Mitsoka's method ("The World of Intestinal Bacteria: Isolation and Identification of Anaerobic Bacteria", 叢 文, 1980). . In addition, nothing was administered to the remaining five human flora mice, and was bred for 4 weeks by the conventional method, and was used as a control.

The following medium was used. In addition, the present inventors prepared the medium without description of a maker name according to the said method of Mitsoka.

1) non-selective badge

a. For anaerobic culture

Easy (EG) agar badge (made by Nissei Seiyakusha), biel (BL) agar badge (made by Nissei Seiyakusha)

b. Aerobic culture

Trypto Soi blood agar medium (Becton Dickinson Corporation)

2) optional badge

a. For anaerobic culture

* BS (A) agar medium (made by Nissei Seiyakusha): Bifidobacterium selective medium

* ES agar badge: Eubacterium selective badge

* NBGT Agar Badge: Bacteroides Selection Badge

* VCS agar badge: Veil1onella selection badge

* NN agar medium: C1ostridium perfringes selection medium

* CCFA agar medium (made by Oxoid): Clostridium difficile selective medium

b. Aerobic culture

* LBD agar medium (Becton Dickinson): Lactobacillus selective medium

* DHL agar medium (made by Nissei Seiyakusha): Enterobacteria selective medium

TATAC Agar Badge: Enterococcus Selective Badge

PEES agar medium: Staphylococcus selection medium

* Potato dextrose agar badge (made by Nisui Seiyakusha): yeast selection badge

* NAC agar medium (made by Nisui Seiyakusha): Pseudomonas aeruginosa selective medium

The sample solution diluted in each said flat medium was dripped, and it was anaerobic incubated for 3 days at 37 degreeC in aerobic culture or anaerobic (Mitsubishi Chemical Co., Ltd. product square type). After incubation, the colonies that appeared were measured, and then gram stained, and bacterial groups were determined from the morphological observation of cells and the presence or absence of colonies and growth under aerobic and anaerobic culture conditions.

3. Test result

The results of this test are as shown in FIG. 8. In the figure, the results of the significant difference test (T test) of both groups are indicated as p <0.05. As is apparent from Fig. 8, No. In the 1088 group from fecal 1g 1.0 × 10 ⁷ ~1O ⁸ of the No. 1088 was detected. In addition, it was confirmed that Bifidobacteria, which are called jade jade bacteria, significantly increased than the control group, and that the rot bacteria (C1ostridia) significantly decreased than the control group. Therefore, No. 1088 has been found to work very favorably on the human intestinal flora.

Summarizing the above test results, as is evident in the in-vitro test shown in Figs. 2, 3, 4, 5 and 6, No. 1088 had a particular inhibitory effect against many pathogens, and its inhibitory effect was remarkable than that of the Lactobacillus johnsoni baseline JCM2012. In addition, in animal experiments, as shown in FIG. 7 and FIG. 8, the inhibitory effect against Helicobacter pylori infection, the increase of bifidus bacteria having a formal action, and the inhibitory effect of rot bacteria were demonstrated. 1088 has proven to be a particularly effective strain as a probiotic.

The present invention is a lactic acid bacterium belonging to the genus Lactobacillus, characterized by having strong acid resistance that can survive for at least 60 minutes in a buffer solution of pH 1.0, proliferation in a short time, and inhibit the growth of pathogens. The present invention relates to a novel lactic acid bacterium, and various products characterized by processing using the novel lactic acid bacteria, and the effects of the present invention are as follows.

1) Since it has excellent acid resistance, it is less killed by gastric acid when ingested or ingested, and provides a new lactic acid bacterium that reaches a large number of bacteria in the intestine and enhances the probiotic effect.

2) This novel lactic acid bacterium can be used for a wide range of products, such as fermented products, health foods, functional foods and pharmaceuticals.

3) This novel lactic acid bacterium has an inhibitory effect of a wide range of pathogens as probiotics, and particularly has a significant inhibitory effect against Helicobacter pylori infection in the stomach.

4) This novel lactic acid bacterium has the effect of increasing the useful bacteria, such as bifidus, and reducing Clostridium, which is a decaying bacterium.

Next, although an Example is described about the most preferable form for implementing this invention, this invention is not limited to a following example.

Example 1

10 liters of VRS broth (Becton Dickinson) No. 1088 was inoculated at a rate of 1 × 10 ⁷ / ml of viable cell concentration, incubated at 37 ° C. for 15 hours, collected after centrifugation, and the collected cells were suspended in sterile saline at a concentration of 0.85%, The washed cells were collected by centrifugation, the washed cells were suspended in a solution containing 10% skim milk powder and 1% sodium glutamate, and lyophilized by a conventional method to prepare about 50 g of dry powder. . It was 2.0 * 10 <^11> / g when the live bacterial count of the obtained dry powder was measured by the same method as the said Experimental example 1.

No. 10 g of dry cells of 1088 were dispersed in 4.99 kg of commercial dextrin (Findex, manufactured by Matsutani Kagakusha Co., Ltd.) having a water content of 3% or less, to prepare about 5 kg of lactic acid bacteria powder. The viable cell count of the obtained lactic acid bacteria powder was measured by the same method as in Test Example 1, and the result was 4.0 × 10 ⁸ / g.

Example 2

To 10 kg of sterilized raw oil, No. 1088, the conventional method, except that an equal amount of mixed bacteria of Lactobacillus bulgaricus (strain isolated from commercial yogurt) and Streptococcus thermophilus (strain isolated from commercial yogurt) was added at a rate of 2%. It fermented in the tank by the tank fermentation type of, and about 10 kg of yogurt was manufactured.

Example 3

No. obtained in Example 1 About 2.0 kg of 1088 dry cells were directly compressed into about 0.3 g, and about 6,500 tablets were produced.

As described above, No. Since 1088 has excellent acid resistance, it can be used for a wide range of products such as fermented products, health foods, functional foods, and pharmaceuticals.

1 shows No. 1088 and Lactobacillus john soni are electrophoretic diagrams of the DNA of LC1.

2 is a Helicobacter pylori No. No. for 13O It is a graph showing the inhibitory effect of 1088.

Figure 3 shows No. It is a graph showing the inhibitory effect of 1088.

Figure 4 shows No. No. for Salmonella thyphimurium LT2. It is a graph showing the inhibitory effect of 1088.

FIG. 5 shows No. 1 for Clostridium diffici1e JCMl296. FIG. It is a graph showing the inhibitory effect of 1088.

FIG. 6 shows No. 1 for Candida a1bicans TI3001. FIG. It is a graph showing the inhibitory effect of 1088.

7 shows Helicobacter pylori No. No. 130 for infected mice. It is a graph showing the inhibitory effect of 1088.

Fig. 8 shows No. 1 of the intestinal flora of human flora mice. A graph showing the effect of 1088.

<110> Yuji AIBA          SNOWDEN KABUSHIKIKAISHA <120> NOVEL LACTIC ACID BACTERIA AND VARIOUS PROCESSED PRODUCTS          PRODUCED BY USING THE LACTIC ACID BACTERIA <130> AIBA-1 <160> 1 <170> KopatentIn 1.71 <210> 1 <211> 1564 <212> DNA <213> Lactobacillus johnsonii No. 1088 <400> 1 gagtttgatc ctggctcagg acgaacgctg gcggcgtgcc taatacatgc aagtcgagcg 60 agcttgccta gatgatttta gtgcttgcac taaatgaaac tagatacaag cgagcggcgg 120 acgggtgagt aacacgtggg taacctgccc aagagactgg gataacacct ggaaacagat 180 gctaataccg gataacaaca ctagacgcat gtctagagtt tgaaagatgg ttctgctatc 240 actcttggat ggacctgcgg tgcattagct agttggtaag gtaacggctt accaaggcaa 300 tgatgcatag ccgagttgag agactgatcg gccacattgg gactgagaca cggcccaaac 360 tcctacggga ggcagcagta gggaatcttc cacaatggac gaaagtctga tggagcaacg 420 ccgcgtgagt gaagaagggt ttcggctcgt aaagctctgt tggtagtgaa gaaagataga 480 ggtagtaact ggcctttatt tgacggtaat tacttagaaa gtcacggcta actacgtgcc 540 agcagccgcg gtaatacgta ggtggcaagc gttgtccgga tttattgggc gtaaagcgag 600 tgcaggcggt tcaataagtc tgatgtgaaa gccttcggct caaccggaga attgcatcag 660 aaactgttga acttgagtgc agaagaggag agtggaactc catgtgtagc ggtggaatgc 720 gtagatatat ggaagaacac cagtggcgaa ggcggctctc tggtctgcaa ctgacgctga 780 ggctcgaaag catgggtagc gaacaggatt agataccctg gtagtccatg ccgtaaacga 840 tgagtgctaa gtgttgggag gtttccgcct ctcagtgctg cagctaacgc attaagcact 900 ccgcctgggg agtacgaccg caaggttgaa actcaaagga attgacgggg gcccgcacaa 960 gcggtggagc atgtggttta attcgaagca acgcgaagaa ccttaccagg tcttgacatc 1020 cagtgcaaac ctaagagatt aggtgttccc ttcggggacg ctgagacagg tggtgcatgg 1080 ctgtcgtcag ctcgtgtcgt gagatgttgg gttaagtccc gcaacgagcg caacccttgt 1140 cattagttgc catcattaag ttgggcactc taatgagact gccggtgaca aaccggagga 1200 aggtggggat gacgtcaagt catcatgccc cttatgacct gggctacaca cgtgctacaa 1260 tggacggtac aacgagaagc gaacctgcga aggcaagcgg atctcttaaa gccgttctca 1320 gttcggactg taggctgcaa ctcgcctaca cgaagctgga atcgctagta atcgcggatc 1380 agcacgccgc ggtgaatacg ttcccgggcc ttgtacacac cgcccgtcac accatgagag 1440 tctgtaacac ccaaagccgg tgggataacc tttataggag tcagccgtct aaggtaggac 1500 agatgattag ggtgaagtcg taacaaggta gccgtaggag aacctgcggc tggatcacct 1560 cctt 1564  

Claims (5)

Lactic acid bacteria belonging to the genus Lactobacillus, the following a) to d): a) having strong acid resistance which survives for at least 60 minutes in a buffer at pH 1.0, b) multiply in a short time, c) inhibiting pathogens, and d) promoting the growth of useful bacteria New lactic acid bacteria, characterized in that having the property of. The method of claim 1, Lactobacillus belonging to the genus Lactobacillus, Lactobaci11us johnsonii (Lactobaci11us johnsonii) No. New lactic acid bacteria, characterized in that 1088 (Accession Number: NITE P-278). Lactic acid bacteria belonging to the genus Lactobacillus, the following a) to d) a) having strong acid resistance which survives for at least 60 minutes in a buffer at pH 1.0, b) multiply in a short time, c) inhibiting pathogens, and d) promoting the growth of useful bacteria Various products characterized in that the processing using a novel lactic acid bacteria having the property of. The method of claim 3, Lactobacillus belonging to the genus Lactobacillus, Lactobacillus Johnsoni No. 1088 (Accession Number: NITE P-278). The method of claim 3, The said processing is fermentation by a novel lactic acid bacterium, the mixing of a novel lactic acid bacterium, the granulation of a new lactic acid bacterium, or the refinement of a new lactic acid bacterium, The various products characterized by the above-mentioned.

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