JP3071128B2 - Lactic acid bacteria isolation and identification method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

This invention relates to a Lactobacillus acidophilus complex.
The present invention relates to a method of culturing a stool sample containing lactic acid bacteria belonging to the L. s complex, selectively separating and cultivating only lactic acid bacteria belonging to the Lactobacillus acidophilus complex, and isolating the same, and a method of identifying and classifying the obtained Lactobacillus acidophilus complex. Further, the present invention provides
It relates to a kit for separating and identifying lactic acid bacteria belonging to such a Lactobacillus acidophilus complex.

[0002]

2. Description of the Related Art Conventionally, the classification of microorganisms has been based on general properties such as morphology and assimilation of sugar. However, the identification method using these phenotypic traits as indicators does not reflect the taxonomic system, There were drawbacks such as low reproducibility of the results and the judgment was easily influenced by the subjectivity of the tester. Therefore, in recent years, a chemical classification method has been used in which bacterial components such as DNA, cell walls, and bacterial proteins derived from bacteria are analyzed by an instrument. Currently, the most important classification method as a taxonomic index is a method for examining DNA homology. Recently, the definition of a microorganism "species" in the taxonomy of microorganisms has also been defined as "a group of strains showing DNA homology of about 70% or more". Furthermore, in microbial taxonomy, phylogenetic taxonomy, which attempts to construct a classification system based on close relationships, has come to dominate.

[0003] Against such a taxonomic background, it is isolated from the human oral cavity, intestinal tract, vagina, or the intestinal tract of pigs, chickens, rats, etc., and settles in the human intestinal tract to host humans. Lactobacillus acidophi (Lactobacillus acidophi)
lus) , and as a result, conventional Lactobacillus acidophilus , Lactobacillus acidophilus (Lactobacillus acidophilus) , Lactobacillus crispatas (Lactobacillus crispatus ) , Lactobacillus amylovoras (Lactobacillus amylovor)
us), Lactobacillus Garinaramu (Lactobacillus
gallinarum ), Lactobacillus gasseri (Lactobacillus
gasseri) , Lactobacillus John Sony (Lactobaci
llus johnsonii)
(Lauer E. et al .: Zbl.Bakt., I.Abt.Orig.C1: 75-78,19
80, Cato EPet al .: International Journal of Syst
ematic Bacteriology, 33: 426-428,1983, Fujisawa T.
et al .: International Journal of Systematic Bacter
iology, 42: 487-491, 1992). In the present invention, these six bacterial species are collectively referred to as Lactobacillus acidophilus complex:
Hereinafter, this is generically referred to as LAC).

[0004] This LAC is called "probiotic"
otics) '', and is a representative species of `` viable microbial additives that benefit host animals by improving the balance of intestinal microbes '' (Fuller R .: Gut, 32: 439-442, 1991, Fu
ller R .: Jurnal of AppliedBacteriology, 66: 365-37
8,1989). Various physiological effects on humans, mice and the like have been recognized in foods to which LAC has been added. In other words, the effect of promoting lactose degradation in the digestive tract
(Kim, HSet al .: Jounal of Dairy Science, 66: 959-96
6,1983), serum cholesterol lowering effect (Gillilan
d, SEet al .: Applied and Environmental Microbiolo
gy, 49: 377-381, 1985), constipation improving effect (Alm L. et al., S
ymposia of Swedish Nutrition Foundation: XV, 131-13
8), anticancer activity (Ayebo, ADet al .: Milchwiss, 35: 730-
733, Goldin, BRet al .: American Journal of Clin.
Nutrition, 39: 756-761, 1984). LAC has also been found to be effective as a feed additive for pigs, chickens and cattle. Examples include improving feed efficiency in piglets (Pollmann, DSet al .: Journal
of Animal Science, 51: 638-644, 1980), reduced incidence of enteritis in piglets (Redmond, HE et al .: Southwest.Vet., 1
8: 287-288, 1965), improving feed efficiency in cattle (Crawford, J.
S. Proceeding 1979 Arkansas Nutrition Conference: 4
5-55), and also improved chicken feed efficiency (Tortuero,
F .: Poult. Science, 52: 197-203, 1973).

[0005] LAC is currently based on the method of Mitsuoka et al.
World of intestinal bacteria: 60-61, 1980, Tomoashi Mitsuoka, Enterobacteriology: 33-
41, 1990). In this method, a modified LBS plate (plate or plate) medium (the composition is shown in Table 1), which is a medium for selecting Lactobacillus, or a non-selective medium for measuring the viable cell count, such as LAC, is used as the separation medium. BL plate and EG plate (compositions are shown in Tables 2 and 3) are used.

[0006]

[Table 1] [How to make a culture medium] After dissolving each component except acetic acid while heating, cool to 50 ° C, add acetic acid and stir.
And There is no need to sterilize the medium.

[0007]

[Table 2] [How to make liver extract] 10g of liver powder with 170ml of purified water
After leaching in a warm bath at 50-60 ° C for about 1 hour, heat at 100 ° C for several minutes, adjust the pH to 7.2, and filter through filter paper. [How to make a culture medium] L-cysteine HCl H ₂ O and components other than horse de-fibrinated blood are dissolved in water, and the pH is corrected. After sterilizing at 115 ° C for 20 minutes, cooling to 50 ° C, L-cysteine HCl H ₂ Add O and horse defibrillated blood, dispense into petri dishes, and plate.

[0008]

[Table 3] Was dissolved by heating the medium Recipe] L-cysteine HCl H ₂ O and components other than horse de繊血Fixed pH, 115 ° C. 20 minutes after sterilization, cooled to 50 ℃, L-cysteine HCl H 2 Add O and horse defibrillated blood, dispense into petri dishes, and plate. As the horse meat leachate, 500 g of horse meat leachate per 1000 ml of medium is used.

When a modified LBS plate is used, a serially diluted sample is applied and cultured under anaerobic conditions free of oxygen. However, when the anaerobic culture is performed on the modified LBS plate, the number of bacteria may appear about 10 ² less than when the anaerobic culture is performed on the BL plate which is a non-selective medium.
Anaerobic cultivation using a BL plate results in the growth of dominant anaerobic bacteria such as Bifidobacterium, Eubacterium, and Bacteroides, in addition to Lactobacillus. And sugar assimilation, growth temperature, etc. must be searched.

[0010]

SUMMARY OF THE INVENTION As described above, LA
C has been confirmed to have many usefulness as a probiotic, and its application is expected. However, no studies on the distribution in the intestinal tract or the like and accurate measurement of the number of bacteria in the intestinal tract have been performed. In addition, the classification of LACs present in the intestinal tract by the new classification system described above has not been sufficiently performed. This is a problem of the conventional separation method,
That is, it is due to low complexity and accuracy and low sensitivity. For example, in the method using the modified LBS plate, Lactobacillus spp. Selectively grows and forms a colony, but it is often observed that the Lactobacillus spp. Appears as a cell count smaller than the actual viable cell count. Further, in order to isolate Lactobacillus spp. Using a non-selective medium, the properties of many colonies must be searched, but this requires enormous manpower and time. Therefore, in order to solve these problems, the selectivity of LAC is higher, and it is possible to omit the trouble of searching for the properties of colonies, assimilation of sugar, growth temperature, and the like. There is a need for a separation method that can reduce the measurement error of the obtained bacterial count.
However, up to the present, analysis of Lactobacillus spp. Has hardly been performed because the above-mentioned problem of the separation method has not been solved. In view of such circumstances, the present inventors have studied a method for efficiently separating LAC and a method for identifying the same. As a result, they have found a method for separating and growing LAC efficiently, and have further found this method and a restriction enzyme specific to lactic acid bacteria. The inventors have found that lactobacilli, particularly LACs, can be separated and identified easily and accurately by combining methods for examining the fragment length, and have completed the present invention. Therefore, an object of the present invention is to provide a method for separating lactic acid bacteria, particularly lactic acid bacteria belonging to LAC among lactic acid bacteria belonging to Lactobacillus, and an identification method using this method. A further object of the present invention is to provide a kit for separating and identifying such lactic acid bacteria.

[0011]

The present invention relates to a Lactobacillus acidophilus complex (Lactobacillusac complex).
In order to isolate and culture lactic acid bacteria belonging to Lactobacillus acidophilus complex from stool samples containing lactic acid bacteria belonging to idophilus complex,
In the medium, the culture is performed under the condition that the oxygen partial pressure during the culture is adjusted to 5 to 15% oxygen concentration, and then the oxygen partial pressure is adjusted to 5 to 15% oxygen concentration in the LBS medium for lactic acid bacteria separation. This is a method of culturing under the conditions described above and isolating microorganisms growing under these conditions as lactic acid bacteria belonging to the Lactobacillus acidophilus complex. Furthermore, DNA derived from lactic acid bacteria specific to lactic acid bacteria belonging to the isolated Lactobacillus acidophilus complex.
The present invention relates to a method for identifying a lactic acid bacterium, which is characterized in that the lactic acid bacterium is identified by whether or not it hybridizes with a restriction enzyme fragment or is identified by a unique restriction enzyme fragment length. Further, the present invention provides a test kit for separating and identifying lactic acid bacteria belonging to Lactobacillus acidophilus complex.

In the method of the present invention, as the first step, LAC is obtained from obligately anaerobic dominant bacterial species such as Bacteroides, Eubacterium and Bifidobacterium.
Perform the following operation to selectively separate The specimen or sample is serially diluted using sterilized physiological saline or sterilized distilled water according to the respective conditions, and applied to an MRS plate. In addition, as a specimen or a sample to which the present method can be applied, food, feces, body fluid, and the like that are expected to contain LAC can be given. However, fecal samples are most desirable,
This operation is a known operation as a usual method for separating lactic acid bacteria. The MRS plate coated with this sample has an oxygen concentration of 5-
Oxygen concentration is adjusted by an anaerobic culture method using a deoxidizer and a carbon dioxide generator (trade name: Anelopack Campiro, manufactured by Mitsubishi Gas Chemical, etc.) in an incubator adjusted to 15% or in a sealable jar. And culture. The MRS medium is a known medium used for the isolation and culture of lactic acid bacteria and has the following composition.

[0013]

[Table 4]

[0014] The oxygen concentration is an oxygen concentration at which obligate anaerobic bacteria cannot grow, which causes measurement errors in the separation of lactic acid bacteria. In the culture of microorganisms, a condition called culture under microaerobic conditions is used. Must be set to By culturing under these conditions, for example, Bacteroides vulgatus (Bacteroides vu) , which is the most dominant bacterial species that becomes an obstacle when isolating lactic acid bacteria present in the intestine from feces etc.
lgatus ), Eubacterium aerofaciens (Eub
Since L. acteriumaerofaciens and Bifidobacterium adolescentis do not grow, lactic acid bacteria, especially LAC, grow selectively. In some samples, cocci are often observed to grow. In this case, the colonies grown on the MRS plate are separated into LB using a known method such as a replica method.
The bacteria that were inoculated into the S medium and grew here can be determined as lactic acid bacteria. This is further collected individually for each colony, identified and classified.

As a method for identifying lactic acid bacteria, identification using DNA is particularly preferred. In the present invention, RFLP (Restriction
Fragment Length Polymorphisms)
(Kamiheiichi et al .: Dairy Sciences and Food Research, 41: A207-210,1
992) or microplate method (Ezaki T. et al .: Internat
ional Journal of Systematic Bacteriology, 39: 224-
229, 1989) for LAC 6 strains. As a method for identifying lactic acid bacteria by the microplate method, a commercially available classification kit such as a trade name: LA plate (manufactured by Snow Brand Milk Products Co., Ltd., sold by Wako Pure Chemical Industries, Ltd.) can be used. Furthermore, identification by the restriction fragment length (RFLP) is accurate and can be performed quickly. The method for separating and identifying lactic acid bacteria of the genus Lactobacillus by RFLP can be easily carried out using a method and a test kit developed by the present inventors. This test method and kit are described in
It is disclosed in JP-A-5-317096. This inspection method is integrated with the present invention by referring to the above publication. In this method, a lactic acid bacterium is easily identified by analyzing and comparing the length of a DNA fragment produced by cleaving a ribosomal RNA gene (rDNA) of a lactic acid bacterium with a specific restriction enzyme. In particular, a specific restriction enzyme is allowed to act on the chromosomal DNA of the test bacterium forming this colony with respect to the colony isolated above,
Identification is performed using the length of the resulting DNA fragment as an index. Such specific restriction enzymes include the restriction enzyme H
in fI and Dra I.

[0016] More specifically, this method firstly involves the use of Lactobacillus acidophilu.
s), Lactobacillus crispatus (Lactobacillus cr
ispatus) , Lactobacillus ga
sseri) and chromosomal DNA are extracted from test bacteria presumed to be contained in the related bacterial species. This extraction is performed by a conventional method of extracting chromosomal DNA, for example, suspending cells in a buffer solution, activating the cells with a lytic enzyme such as N-acetylmuramidis SG, lysing the cells, repeating phenol treatment and ethanol precipitation. By dissolving the obtained precipitate in water, a chromosomal DNA solution can be obtained. This chromosomal DNA was directly digested with restriction enzymes Hin fI and Dra I, subjected to agarose gel electrophoresis, and labeled with a radioisotope or a non-radioactive 16 SrDN.
The cleavage fragment is identified by Southern hybridization using A as a probe. Alternatively, 16S rDNA is extracted from the extracted chromosomal DNA by enzymatic gene amplification.
Specifically amplified by the restriction enzymes Hin fI and D
Cleavage with ra I and running on agarose gel electrophoresis. The lengths of the cut DNA fragments obtained by these methods are compared with those of the standard strain, and the test bacteria showing an electrophoretic pattern consistent with the standard strain are assumed to be included in the same species as the standard strain. Any restriction enzyme can be used as long as it specifically cleaves rDNA of a lactic acid bacterium and exhibits a pattern peculiar to the species during electrophoresis. In this respect, the restriction enzymes Hin fI and Dra I are used in Lactobacillus acidophilu.
s), Lactobacillus crispatus (Lactobacillus cr
ispatus) , Lactobacillus ga
sseri) and its relatives are selected as optimal restriction enzymes for cleaving rDNA. However, combinations of restriction enzymes other than these may be used as long as they exhibit an rDNA migration pattern specific to the bacterial species. Thus, lactic acid bacteria belonging to LAC can be easily and quickly separated and identified.

The kit for separation and identification of the present invention comprises: (1) an MRS plate for smearing a subject; (2) an LBS plate for transplanting colonies grown on the MRS plate; and (3) microaerobic culture. It is composed of an oxygen scavenger and a carbon dioxide generator to be performed, and (4) an identification reagent using DNA. For identification by DNA, an identification means using a microplate or an identification means by RFLP can be exemplified. Among them, a method of classifying and identifying lactic acid bacteria of the genus Lactobacillus by RFLP will be described as an example. The identification reagent is composed of a reagent for extracting DNA from the test cells, a reagent for specifically amplifying the ribosomal RNA gene of lactic acid bacteria, restriction enzymes Hin fI and Dra I, a reaction reagent, and a standard strain as a control sample. Of the cleaved DNA or data related thereto. Examples of the reagents include enzyme solutions and surfactants required for lysis, and phenol and ethanol required for chromosomal DNA purification.
Contains two primers specific to the 16S ribosomal gene, in addition to the heat-resistant DNA synthase, buffer, and substrate required for the enzymatic gene amplification method. Contains a restriction enzyme and a reaction buffer. As the reagents, a cut DNA fragment of a standard strain which can be compared and confirmed by performing electrophoresis in parallel with a test sample at the time of electrophoresis performed in the final stage of identification work, or a generally available DNA
A diagram showing the relationship between the size marker and the cut DNA fragment of the standard strain is included. The kit for classification of lactic acid bacteria of the present invention is provided by using a kit composition comprising only the culture reagent configured as described above or a combination of the culture reagent and an identification method using DNA.

Hereinafter, Test Examples and Examples serving as the basis of the present invention will be shown, and the present invention will be described in more detail.

[Test Example 1] In this test example, lactic acid bacteria present in human feces and dominant anaerobic bacteria groups such as Bifidobacterium, Eubacterium, and Bacteroides that hinder the isolation of the lactic acid bacteria were determined. It shows that it is possible to selectively separate and culture by adjusting the oxygen partial pressure during culture. Human predominate bacterium in feces Bacteroides Val <br/> Gatasu (Bacteroides vulgatus), Eubacterium Aero tumefaciens (Eubacterium aerofaciens), Bifidobacterium address Sen infantis (Bifidobacterium
adolescentis ) and Lactobacillus acidophilus , Lactobacillus gasseri , which are typical bacteria of LAC, were tested for growth by oxygen partial pressure using Lactobacillus gasseri as test bacteria. In culturing, the above BL plate, MRS plate, LBS pla
(1) Oxygen partial pressure 10%, carbon dioxide partial pressure 10
%, (2) oxygen partial pressure 0%, carbon dioxide partial pressure 20%. The oxygen partial pressure was adjusted using a commercially available aneropack obtained by combining a commercially available oxygen absorber for anaerobic culture and a carbon dioxide generator. Product name: Aneropack /
Campiro (Mitsubishi Gas Chemical Co., Ltd.) was used, and under the conditions of (2), the product name: Anelopack Kenki (Mitsubishi Gas Chemical Co., Ltd.) was used. Each microorganism was diluted to 10 ⁹ / ml with physiological saline, applied to each 1 ml plate, and cultured at 37 ° C. for 48 hours at each oxygen partial pressure and carbon dioxide partial pressure, and the number of generated colonies was counted. The bacterial count was determined. The measurement results are shown in FIG. As is apparent from these results, Bacteroides, which is an obstacle to LAC isolation, when cultured on LBS or MRS plates under conditions of 10% oxygen partial pressure and 10% carbon dioxide partial pressure when cultured under conventional anaerobic conditions.・
Vargatus (Bacteroides vulgatus ), Eubacterium aerofaciens (Eubacterium aerofaciens) ,
Bifidobacterium adressentis
rium adolescentis ) does not grow at all,
It was confirmed that AC could be measured.

[0019]

[Test Example 2] In this test example, human feces were used as a sample, and the oxygen partial pressure 10% and carbon dioxide partial pressure 1 confirmed in Test Example 1 above.
An example is shown in which LAC was measured under 0% conditions and it was confirmed that accurate LAC measurement could be performed. Human feces are suspended in physiological saline, and this is used as a sample on an MRS plate at an oxygen partial pressure of 1%.
The number of colonies generated by culturing at 0%, carbon dioxide partial pressure of 10% and culturing at 37 ° C. for 48 hours was counted. Similarly dilutions of Lactobacillus gasseri (Lactobacillus gasseri), and with, the fecal dilutions Lactobacillus gasseri (Lactoba
The same measurement was performed for a mixed solution of a diluted solution of Cillus gasseri) . In addition, the final dilution of the mixed solution of the stool diluent and the diluent of Lactobacillus gasseri (Lactobacillus gasseri) is the stool dilution or Lactobacillus gasseri.
(Lactobacillus gasseri) was prepared by mixing an equal volume of a two-fold concentration diluent so as to have the same dilution ratio as the diluent.
The number of generated colonies is shown in Table 5 below.

[0020]

[Table 5] 試 料 Sample Number of generated colonies ────────────希 釈 Stool diluent 2185 Lactobacillu s gasseri diluent 2215 Stool diluent + Lactobacillu s gasseri diluent 4423 ──────────── ──────────────────

Stool dilution + Lactobacillus gasseri (Lac
(Tobacillus gasseri) The number of colonies of the diluted solution is the fecal diluted solution and Lactobacillus gasseri (Lactobacillus gasseri)
It almost coincided with the sum of the number of colonies of each dilution. This is due to the fact that Lactobacillus ga
sseri) means that they can be separated exactly. Therefore, LA can be easily obtained by this culture method.
It became clear that C could be separated.

[0022]

Example 1 This example shows an example in which lactic acid bacteria were separated and cultured from human feces by the method of the present invention and identified by the RFLP method. Stool was collected from volunteer A who had not eaten fermented food for one month, 1 g of this was collected, serially diluted with physiological saline, applied to an MRS plate, and treated in the same manner as in the above Test Example 1 (1). 10% oxygen concentration
The cells were cultured at 37 ° C. for 48 hours under slightly aerobic conditions. Among the grown plates, the serial dilution ratio was 2 × 10 ⁴ dilution 1
165 colonies grown on one MRS plate were inoculated on an LBS plate by a replica method. When this was cultured at 37 ° C. for 48 hours under microaerobic conditions of about 10% oxygen concentration, 5 strains of bacilli grew. These five strains of bacilli were disclosed in
It was identified by the RFLP method according to the method using the standard strain described in the Examples disclosed in Japanese Patent No. 317096. As a result, two of the five strains were Lactobacillus acidophilus (Lactobacill).
us acidophilus) . Therefore, it was found that the detection level of LAC in feces of this volunteer A was 4 × 10 ⁴ cfu. The time required for the above inspection was 6 days including the separation culture. On the other hand, the same sample is used in the same manner as before using a BL plate,
The usual method of separating LAC, that is, anaerobic cultivation, and identification of the generated colonies by a biochemical method required an inspection period of one month or more. LAC could not be detected. This test makes it possible to determine whether or not useful LACs are resident in the intestine, and can evaluate the effects of eating.

[0023]

[Example 2] As in Example 1, feces were collected from volunteer K who had not eaten fermented food for one month, serially diluted with physiological saline, and applied to an MRS plate. The cells were cultured at 37 ° C. for 48 hours under microaerobic conditions with an oxygen concentration of about 10%. When 250 colonies generated from 10 MRS plates with a serial dilution ratio of 1 × 10 ⁵ out of the grown plates were inoculated into LBS broth, 28 strains of bacilli grew. Example 1
As a result of identification by the RFLP method, it was found that two strains were Lactobacillus gasseri . In this case, the detection level of LAC was 2 × 10 ⁴ cfu / g of feces. The time required for the above inspection was 5 days including the separation culture. The detection level per stool is said to be undetectable with a conventional technique at a detectable level of 10 ⁶ cfu or less, and it has been clarified that detection can be performed at a level much lower than this level. Therefore, the effect of increasing LAC by a meal or the like can be determined at an early stage.

[0024]

Example 3 This example shows the construction of a kit for LAC separation and identification which can be used when feces are used as a sample. Composition of 10-sample kit (1) 10 MRS plates (2) 10 LBS plates (3) 10 packs of oxygen scavenger (4) 10 packs of carbon dioxide generator (5) LAC RFLP detection reagent 16S rDNA specifically Lactobacillus acidophilus by the method of amplification (Lactobacillus acidophilus) ,
Lactobacillus crispatus (Lactobacilluscrispatu
s) , Lactobacillus gasseri
And the reagent for identifying the related bacterial species are composed of the following components (for 10 samples).

Reagent for extracting DNA from cells Enzyme reaction buffer [Tris-maleic acid buffer, pH 6.5, 1M saccharose] 40 ml Lysis enzyme solution [N-acetylmuramide SG] 200 μl 10% SDS solution 300 μl Phenol mixture [ Phenol: chloroform: isoamyl alcohol = 25: 24: 1] 5ml ethanol 10ml 70% ethanol 5ml Washing solution [0.1xSSC (SSC: 0.15M NaCl, 0.015M trisodium citrate solution)] 2.5ml To specifically amplify 16S rDNA Reagent Taq DNA polymerase 25 Units 10 × Enzyme reaction buffer [100 mM Tris-HCl, pH 8.3, 500 mM KCl, 15 mM MgCl ₂ ] 100 μl 10 × dNTP mixture [2 mM dATP, dCTP, dGTP, dTTP] 100 μl Primer 1 [5 ′ -AGAGTTTGATCCTGGCTCAG-3 '] 100 pmole Primer 2 [5'-AAGGAGGTGATCCAGCCGCA-3'] 100 pmole Reagent for cleaving 16S rDNA Restriction enzyme Hin fI (5 Unit / μl) 10 μl 10 × Hin fI reaction buffer [50 mM Tris-HC l, pH 7.5, 10 mM MgCl ₂ , 1 mM DTT, 100 mM NaCl] 20 μl Restriction enzyme Dra I (5 Unit / μl) 10 μl 10 × Dra I reaction buffer [10 mM Tris-HCl, pH 7.5, 10 mM MgCl ₂ , 1 mM DTT, 50mM NaCl] 20μl

Standard sample Hin fI standard sample for homology group A1 (1 mg DNA / ml) 20 μl Dra I standard sample for homology group A1 (1 mg DNA / ml) 20 μl Hin fI standard sample for homology group A2 (1 mg DNA / ml) 20 μl Dra I standard sample for homology group A2 (1 mg DNA / ml) 20 μl Hin fI standard sample for homology group A3 (1 mg DNA / ml) 20 μl Dra I standard sample for homology group A3 (1 mg DNA / ml) 20 μl for homology group A4 Hin fI standard sample (1 mg DNA / ml) 20 μl Dra I standard sample for homology group A4 (1 mg DNA / ml) 20 μl Hin fI standard sample for homology group B1 (1 mg DNA / ml) 20 μl Dra I standard sample for homology group B1 ( 1mg DNA / ml) 20μl Hin for Moroji Group B2 fI standard sample (1mgDNA / ml) Dra for 20 [mu] l homology group B2 I standard sample (1mgDNA / ml) 20μl standard samples were prepared from a standard strain is shown in Japanese Patent Laid-Open No. 5-317096 .

[0027]

Example 4 This example shows the construction of a kit for LAC separation and identification that can be used when feces are used as a sample. Composition of 10 sample kits (1) 10 MRS plates (2) 10 LBS plates (3) 10 packs of oxygen absorber (4) 10 packs of carbon dioxide generator (5) LLP RFLP detection reagent Biotin-labeled RNA probe Lactobacillus acidophilus (Lactobacillus acidophilus) ,
Lactobacillus crispatus (Lactobacilluscrispatu
s) , Lactobacillus gasseri
And a kit for identification of related bacterial species are composed of the following components (for 10 samples).

Reagent for extracting DNA from bacterial cells Enzyme reaction buffer (Tris-maleic acid buffer, pH 6.5, 1M saccharose) 40 ml Lysis enzyme solution [N-acetylmuramidase SG] 200 μl 10% SDS solution 300 μl Phenol mixture 5 ml [Phenol: chloroform: isoamyl alcohol = 25: 24: 1] Ethanol 10ml 70% ethanol 5ml Washing solution [0.1xSSC (SSC: 0.15M NaCl, 0.015M trisodium citrate solution)] 2.5ml Reagent for cleaving 16S rDNA Enzyme Hin fI (5 Unit / μl) 10 μl 10 × Hin fI reaction buffer [50 mM Tris-HCl, pH 7.5, 10 mM MgCl ₂ 20 μl 1 mM DTT, 100 mM NaCl] Restriction enzyme Dra I (5 Unit / μl) 10 μl 10 × Dra I reaction buffer [10mM Tris-HCl, pH 7.5,10mM MgCl 2, 1mM DTT, 50mM NaCl] reagents Lactobacillu s acidophilus type strain (JCM1132) for making 20μl nonradioactive labeled RNA probe 16 SrRNA 100μg photo biotin Or photoprobe biotin (1 mg / ml) 100 μl 0.1 M Tris-HCl (pH 9.0) buffer 1 ml 2-butanol 1 ml Reagent for detecting non-radiolabeled substance streptavidin-alkaline phosphatase (1 mg / ml) 70 μl Nitroblue tetrazolium (NBT) (75 mg / ml) 500 μl 5-Bromo-4-chloro-3-indole phosphate (BCIP) (50 mg / ml) 500 μl

Standard sample Hin fI standard sample for homology group A1 (1 mg DNA / ml) 20 μl Dra I standard sample for homology group A1 (1 mg DNA / ml) 20 μl Hin fI standard sample for homology group A2 (1 mg DNA / ml) 20 μl Dra I standard sample for homology group A2 (1 mg DNA / ml) 20 μl Hin fI standard sample for homology group A3 (1 mg DNA / ml) 20 μl Dra I standard sample for homology group A3 (1 mg DNA / ml) 20 μl for homology group A4 Hin fI standard sample (1 mg DNA / ml) 20 μl Dra I standard sample for homology group A4 (1 mg DNA / ml) 20 μl Hin fI standard sample for homology group B1 (1 mg DNA / ml) 20 μl Dra I standard sample for homology group B1 ( 1mg DNA / ml) 20μl Hin for Moroji Group B2 fI standard sample (1mgDNA / ml) Dra for 20 [mu] l homology group B2 I standard sample (1mgDNA / ml) 20μl standard samples were prepared from a standard strain is shown in Japanese Patent Laid-Open No. 5-317096 .

[0030]

According to the practice of the present invention, lactic acid bacteria, in particular,
Provided is a method for separating lactic acid bacteria classified as LAC among lactic acid bacteria belonging to the genus Lactobacillus, and an identification method using this method. In particular, the present invention enables rapid separation of LACs, which has conventionally required a lot of manpower and time. According to the separation method of the present invention, lactic acid bacteria,
In particular, since LAC can be rapidly separated, this method can be used for searching for a useful strain, testing the cause of abnormal fermentation of beer, and the like. Further, the lactic acid bacteria thus separated can be identified quickly and accurately. In particular, LAC
This identification method can be used for clinical tests, process control, and the like, because the detection sensitivity can be increased to about 100 to 1000 times. Therefore, the separation and identification kit of the present invention is useful in these fields.

[Brief description of the drawings]

FIG. 1 Bacteroides vulgatus , a dominant bacterium in human feces, performed in Test Example 1.
, Eubacterium aerofaciens
maerofaciens), Bifidobacterium address first move chair (Bifidobacteriumadolescentis) and Lactobacillus, which is a typical bacteria LAC acidophilus (Lactobaci
llus acidophilus) , Lactobacillus gasseri (Lactoba
FIG . 3 shows the results of a test in which the effect of growth due to oxygen partial pressure was measured using cillus gasseri) as a test bacterium.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification code FI C12R 1:23) (C12N 1/20 C12R 1: 225) (C12Q 1/04 C12R 1:23) (C12Q 1/04 C12R 1 : 225) (56) References JP-A-5-317096 (JP, A) JP-B-47-33148 (JP, B1) of Dairy Science, 1988, Vol. 71, No. 12, p. 3222-3229 (58) Fields investigated (Int. Cl. ⁷ , DB name) C12N 1/20 C12Q 1/02 BIOSIS (DIALOG) WPI (DIALOG)

Claims (4)

(57) [Claims] 1. A lactic acid bacteria-containing fecal sample belonging to the Lactobacillus acidophilus complex was adjusted to have an oxygen partial pressure of 5 to 15% during culturing in an MRS medium for culturing and separating lactic acid bacteria. Culture under conditions, then L for lactic acid bacteria isolation
Lactobacillus acidophilus complex (Lactobacillus complex) characterized in that culturing is performed under conditions adjusted to an oxygen partial pressure of 5 to 15% in a BS medium, and only lactic acid bacteria are selectively grown and separated.
acidophilus complex). 2. A lactic acid bacteria-containing fecal sample belonging to the Lactobacillus acidophilus complex was adjusted to have an oxygen partial pressure of 5 to 15% during culture in an MRS medium for lactic acid bacteria separation and culture. Culture under conditions, then L for lactic acid bacteria isolation
Lactobacillus acidophilus characterized by being cultured in a BS medium under conditions adjusted to an oxygen partial pressure of 5 to 15%, selectively growing only lactic acid bacteria, separating and identifying the lactic acid bacteria. Complex (Lactobaci
llus acidophilus complex). 3. The separation and identification method according to claim 2, wherein the identification is performed based on whether or not hybridization occurs with a specific DNA restriction enzyme fragment derived from a lactic acid bacterium, or by comparing the specific restriction enzyme fragment length. 4. An MRS medium and an LBS medium, a deoxygenating agent capable of adjusting the oxygen partial pressure of a closed container for lactic acid bacteria culture to 5 to 15%, a DNA hybridization reagent for identifying lactic acid bacteria or a restriction enzyme specific to lactic acid bacteria. 4. A separation and identification kit for performing the method for separating and identifying lactic acid bacteria according to claim 3, comprising a fragment length determination reagent.