JPH11332593A - Detection of coliform group bacterium - Google Patents

Abstract

PROBLEM TO BE SOLVED: To rapidly detect a coliform group bacterium in foods, cooking utensils, etc., in order to prevent putrefaction of foods or food poisoning by measuring a β-galactosidase of the coliform group bacterium using a luciferin-O- galactoside and luciferase. SOLUTION: 2-Cyano-6-methoxybenzothiazole is demethylated according to a conventional method to provide 2-cyano-6-hydroxybenzothiazole, which is then reacted with 2,3,4,6-tetra-O-acetyl-α-D-galactopyranosyl bromide, glycosylated, then condensed with D-cysteine and further deacetylated to thereby afford a luciferin-O-galactoside. A β-galactosidase of the coliform group bacterium in a sample subjected to the selective culture of a Gram-negative bacterium is measured by using the luciferin-O-galactoside and luciferase to rapidly detect the coliform group bacterium in the sample collected from foods, cooking utensils, etc.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a method for detecting coliform bacteria.

[0002]

2. Description of the Related Art The group of coliforms is a group of aerobic or facultative anaerobic gram-negative spore-free bacilli that produce acid and gas by decomposing lactose (Food Sanitation Guidelines).
The coliforms are considered an indicator of fouling and are desirably less in food and cooking utensils. As a simple method for detecting coliforms, β-galactosidase (hereinafter abbreviated as β-gal) of a gram-negative bacterium that has been selectively cultured and grown.
Is widely used. At present, a chromogenic substrate or a fluorescent substrate is used to detect β-gal in the coliform group (Journal of the Japan Society for Food Microbiology, Vol. 14, 149-154).
P. 1998). These methods require about 18 hours or more to detect coliforms.

[0003]

SUMMARY OF THE INVENTION In order to prevent spoilage and food poisoning of foods, it is desirable to more rapidly detect coliform bacteria in foods and cooking utensils. Therefore, it is not always appropriate that the current detection time is about 18 hours or more. The present invention is directed to a method for detecting Escherichia coli that selectively cultures gram-negative bacteria and detects β-gal of the grown bacteria, thereby improving the method and shortening the detection time. Generally, the luminescence method has better sensitivity than the color development method or the fluorescence method.

[0004]

Means for Solving the Problems Accordingly, the present inventors have
Using a compound of beetle-derived luciferin and galactoside as a luminescent substrate for β-gal, we attempted to develop a rapid detection system for coliform bacteria that detects luciferin generated by the action of β-gal with beetle-derived luciferase. Thus, the present invention has been completed. That is, the present invention is a method for detecting Escherichia coli, which comprises measuring β-galactosidase of Escherichia coli using luciferin-O-galactoside and luciferase.

[0005]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below in detail.
First, a microorganism is transferred to a medium from an object to be inspected, for example, food or cooking utensil, and cultured. The food may be inoculated directly into the medium, or microorganisms may be extracted from the food. To extract microorganisms from food, a stomacher may be used. When wiping microorganisms from a cooking utensil or the like, a cotton swab impregnated with physiological saline or a buffer can be used. The culture medium for culturing the microorganism may be any medium as long as it allows selective growth of Gram-negative bacteria. For example, a nutrient broth to which about 0.01% of sodium lauryl sulfate or about 0.1% of sodium deoxycholate is added. Also, by adding about 1 mM isopropyl-β-thiogalactoside (hereinafter abbreviated as IPTG) or about several% lactose to the medium, it is possible to increase the β-gal production of the microorganism. The culturing temperature is about 15 ° C to 45 ° C, preferably about 30 ° C to 40 ° C. It may be ventilated or sealed, and may be shaken if necessary.

[0006] The culturing time is 3 to 48 hours, preferably about 4 to 24 hours. Next, the cultured microorganism is crushed, and β-gal contained in the cells is extracted. The crushing operation may be performed by any method as long as the β-gal activity is not impaired, and can be performed by, for example, ultrasonic treatment, treatment with a lytic enzyme or a lytic agent, French press, or the like. The β-gal of the cell lysate is measured by the following method, and if the activity is observed, it can be determined that the coliforms are present in the inspected object, for example, food or cooking utensil.

The present invention provides a β-gal substrate, for example,
D-Firefly luciferin-O-β-D-galactopyranoside sodium salt (hereinafter abbreviated as L-Gal)
No. 71 publication. ) Is used. When a compound of beetle luciferin and galactoside is added to the cell lysate, luciferin is produced if β-gal is present in the cell lysate. If a luminescent reagent containing luciferase, ATP, and magnesium is added to the reaction solution, which includes natural or recombinant beetles (including modified types such as mutant types), luciferin is decomposed and emits light. . The beetle luciferase includes, for example, firefly luciferase, click beetle luciferase, and the like. The luminescent reagent includes a protein (for example, bovine serum albumin), a reducing reagent (for example, β-mercaptoethanol), a chelating agent [for example, ethylenediaminetetraacetic acid (hereinafter, abbreviated as EDTA) for stabilizing luciferase activity. ] May be added. Β-gal activity can be measured by measuring the luminescence reaction using, for example, a luminometer or the like.

[0008]

EXAMPLES The present invention will be described more specifically with reference to the following examples. Example 1 Production of L-Gal 4.4 g (23 mmol) of 2-cyano-6-methoxybenzothiazole (obtained from Wako Pure Chemical Industries) was added to Bull. Chem. Soc. Jpn., 65.
(1992) Demethylation was carried out according to the method of 392-395 to give 2-
2.8 g (69% of theory) of cyano-6-hydroxybenzothiazole were obtained. Next, Carbohydrate Research, 205 (1
990) 2.8 g (16 mmol) of 2-cyano-6-hydroxybenzothiazole and 2,3,4,6-tetra-O-acetyl-α-D-galactopyranosyl bromide (obtained from Sigma) according to 225-233 ) From 10.6 g (28 mmol) by glycosylation to 2
2.6 g of -cyano-6- (2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyloxy) benzothiazole (theoretical
44%), of which 2.6 g (5 mmol) was condensed with D-cysteine hydrochloride, followed by deacetylation to give L-Gal 1.0 g.
(42% of theory).

[0009] 2. Measurement of β-gal using L-Gal As a β-gal measurement reagent containing L-Gal, luciferase and ATP, a reagent having the following composition was prepared [0.5 mM ATP, 50 mM
Tricine, 1 mM EDTA, 10 mM magnesium acetate, 1 mM dithiothreitol (DTT), 0.2% bovine serum albumin (BSA), 30 μ
g / ml Genji firefly luciferase (manufactured by Kikkoman), 70 μg / ml L-Gal, pH 7.8]. After preparation, the β-gal measurement reagent was left at 25 ° C. for 3 hours, and the integrated luminescence amount for 10 seconds was 3,000 counts or less (Lumitester K manufactured by Kikkoman Co., Ltd.).
Use -200. ). Enzyme diluent [50
E. coli-derived β-gal (manufactured by Oriental Yeast) was diluted with mM Tricine, 1 mM EDTA, 1 mM DTT, 0.2% BSA, pH 7.8],
^{10 -7, 10 -6, 10 -5} , 10 -4, 10 -3, 10 - 2, 10 -1 IU / ml of diluted solution was prepared. 5 μl of β-gal diluted solution of each concentration was added to 100
It was added to μl of the β-gal measurement reagent, and the change over time in the amount of luminescence was measured using Lumitester K-200 (manufactured by Kikkoman). A case where the amount of luminescence increased by 200 counts or more per minute was judged as positive, and a case where the count increase per minute was less than 50 was judged as negative. 1
A case where the count increase per minute was 50 or more and less than 200 was regarded as a false positive. As a control experiment, a fluorescent substrate (4-methylumbelliferyl
-β-galactoside (4-MU-Gal)], and the activity of the β-gal diluted solution was measured according to an enzyme immunoassay (P. Tijssen, translated by Eiji Ishikawa, Tokyo Chemical Dojin, p332-336). As a result, β-ga when L-Gal and 4-MU-Gal were used
l detection sensitivity (the concentration of the diluent added) are each 10 ^{- 5} and 1
0 ^- it was ⁴ IU / ml. That is, the luminescence method using L-Gal was found to be 10 times more sensitive than the fluorescence method using 4-MU-Gal. In addition, the fluorescence method has an advantage that the result can be determined in about 5 minutes, while the luminescence method requires about 50 minutes for measurement.

[0010] 3. Measurement of coliform bacteria using L-Gal Escherichia coli 1100 (obtained from Max-Planck Institute, Heidelberg, Germany) was cultured overnight in a nutrient broth manufactured by Oxoid, then diluted with physiological saline, and the same medium +
1 mM IPTG (β-gal inducer manufactured by Wako Pure Chemical Industries) in 50 colony forming units (hereinafter abbreviated as CFU) / m
Inoculated so as to obtain l. The culture was allowed to stand still at 37 ° C. in a closed state, and samples were taken over time from the 4th hour after the start of the culture to the 8th hour every hour, and the cells were disrupted by ultrasonic treatment. The β-gal activity of the crushed liquid was measured by the luminescence method using L-Gal and the fluorescence method using 4-MU-Gal in the same manner as described above. The results showed that the luminescence method could detect up to 6 hours of culture of E. coli (2.0 × 10 ⁴ CFU / ml), while the fluorescence method could detect only 7 hours of culture of E. coli (1.4 × 10 ⁵ CFU / ml). Did not. That is, it was revealed that the luminescence method using L-Gal has higher sensitivity than the fluorescence method using 4-MU-Gal even when measuring Escherichia coli.

4. Examination of selectivity It was found that E. coli can be measured with high sensitivity by the luminescence method using L-Gal. In addition, it was confirmed that strains belonging to the Escherichia coli group other than Escherichia coli could be measured, and in combination with the selective culture, the results were negative for microorganisms other than the Escherichia coli group. Citrobacterfreundii (ATCC 8090), Klebsiella pneu
moniae (ATCC 13883) (E. coli group), Salmonella Typ
himurium (ATCC 29629), Morganella morganii (ATCC 2583
0), Proteus mirabilis (ATCC 29906), Pseudomonas aerug
inosa (ATCC 10145) (Gram-negative bacteria other than coliforms), Micrococcus luteus (ATCC 4698), Enterococcus f
aecalis (ATCC 19433), Streptococcus agalactiae (ATCC
13813), Corynebacterium renale (ATCC 19412), Bacillus
subtilis (ATCC 6051), Staphylococcus aureus (ATCC126
00) (the above Gram-positive bacteria) were cultured overnight in Nutrient Broth + 1 mM IPTG manufactured by Oxoid, and the cells were disrupted by sonication. The β-gal activity of these strains was measured using the L-Gal reagent in the same manner as above, and all were positive in the coliform group, all negative in gram-negative bacteria other than the coliform group, and all positive in gram-positive bacteria. . That is, when selective culture was not performed, β-gal activity was found in coliform bacteria and Gram-positive bacteria. Next, 0.1% in the above medium
Sodium deoxycholate was added, only gram-negative bacteria were selectively cultured overnight, and β-gal activity was measured in the same manner.
As a result, only the coliform group was positive, and all Gram-positive bacteria were negative. From the above results, the combination of selective culture of Gram-negative bacteria and the method of measuring β-gal activity using L-Gal enabled the specific detection of Escherichia coli.

[0012]

According to the present invention, the detection time can be shortened in the method for detecting Escherichia coli group in which gram-negative bacteria are selectively cultured and β-gal of the grown bacteria is detected, and the present invention is useful. It is.

Continuation of the front page (72) Inventor Atsushi Sano 339 Noda, Noda City, Chiba Prefecture Kikkoman Corporation

Claims (3)

[Claims] 1. A method for detecting coliform bacteria, which comprises measuring β-galactosidase of coliforms using luciferin-O-galactoside and luciferase. 2. The method for detecting Escherichia coli according to claim 1, wherein the selective cultivation is carried out prior to the measurement of β-galactosidase of the Escherichia coli. 3. The method for detecting coliform bacteria according to claim 2, wherein the selective culture is a selective culture of gram-negative bacteria.