JPH10191996A - Method for measuring concentration of ATP derived from microorganisms in sample - Google Patents

Abstract

(57) Abstract: A method for selectively detecting ATP derived from microbial cells from a sample containing a mixture of microbial cells and non-microbial cells. SOLUTION: In a sample containing a mixture of microbial cells and non-microbial cells, a step of mixing a nonionic surfactant to bring out ATP of the non-microbial cells outside the cells, centrifuging the sample to remove a supernatant, and removing microorganisms Step of forming cell pellet, adding buffer to microbial cell pellet, pH = 6.0
8.0 and a protease-free soluble protein, A
Adding a TP hydrolase and an ATP hydrolase activator, adding an ATP hydrolase inhibitor,
A method of extracting ATP from microbial cells by adding an ATP extractant; and a step of measuring the concentration of ATP released from the microbial cells using a bioluminescence method. .

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a microbiological quality inspection of a sample which is a mixture of microbial cells and non-microbial cells.
The present invention relates to a method for simply and selectively measuring P in a short time.

[0002]

[Prior art]

[0003] A bioluminescence method using firefly luciferase is widely used as a rapid detection method for ATP of microbial cells. This method has become popular as a method for measuring microbial cell-derived ATP in a sample that is mainly a mixture of microbial cells and non-microbial cells. However, since ATP to be measured is widely present in the biological world, Various devices are required to selectively detect only the target microbial cell-derived ATP.

[0004] Various methods have been developed as methods for selectively measuring ATP derived from microbial cells in a sample containing non-microbial cells. In any case, the method of detecting only the microorganism-derived ATP selectively from the large amount of non-microbial cell-derived ATP with high sensitivity is devoted to the method. inside that,
AT derived from non-microbial cells by non-ionic surfactant
After extracting P, it is decomposed with an ATP hydrolase such as apyrase, and then the ATP derived from microbial cells is extracted and measured by a bioluminescence method. This is an effective means for improving the selective detection sensitivity. On the other hand, however, there are disadvantages that arise from using ATP hydrolase. That is, after decomposing ATP derived from non-microbial cells, ATP hydrolyzing enzyme remaining in the sample may degrade even ATP derived from microbial cells of interest. Therefore, in the method using ATP hydrolase, in any case, various measures have been developed by devising the treatment of ATP hydrolase.

That is, US Pat. No. 3,745,090 describes several methods for inactivating apyrase, which is an ATP hydrolase. Addition of acids, bases, heavy metals, organic solvents, enzyme inhibitors as chemical methods, heating and use of short wavelength radiation as physical methods have been suggested, among which heat inactivation with boiling Tris-EDTA buffer. However, this method involves nonspecific and irreversible denaturation of all proteins, including microbial cells of interest, and measurement sensitivity due to dilution of the sample with boiling Tris-EDTA buffer. Drops. The patent also suggests the use of an enzyme activity inhibitor, but does not mention a specific method.

[0006] Separately, a method has been developed in which ATP hydrolase is used at a very low concentration so that the desired ATP is not rapidly decomposed (US Pat. No. 430).
No. 3752). However, in this method, it is necessary to severely restrict the use conditions of the ATP hydrolase, and
Due to the slow action of the hydrolase, it took a considerable amount of time to sufficiently decompose the non-target ATP, which was the measurement background.

Japanese Patent Publication No. Sho 62-4120 discloses a method of hydrolyzing ATP derived from non-microbial cells with an ATP hydrolase such as apyrase, and then passivating the apyrase with glass beads or the like. But with this method,
High sensitivity detection cannot be expected because the sample is not subjected to the operation of concentrating microbial cells, or is subject to measurement inhibition by sample components such as protein and milk fat. Further, there is a problem that the removal or inactivation of apyrase is incomplete, and even after ATP is extracted from the microbial cells, the apyrase has an inhibitory effect on apyrase and an accurate measurement value cannot be obtained. Therefore, large variation in the measurement values, it is practically ^{10 6 CFU / ml (CFU;} C olony F ormi
ng U nit) about the detection sensitivity only can not be expected.

[0008]

In view of the above facts, the present invention provides a method for selectively measuring ATP derived from microbial cells in a sample which is a mixture of microbial cells and non-microbial cells, using ATP hydrolase. In addition, the present invention provides a method for simply and highly sensitively measuring only microbial cell-derived ATP by quickly inactivating ATP hydrolase so as not to cause decomposition of the target microbial cell-derived ATP. It is in.

[0009] The present inventors have found conditions for effectively deactivating non-microbial ATP by effectively using ATP hydrolase, and quickly inactivating ATP hydrolase after hydrolysis. It has become possible to selectively and highly sensitively detect only ATP derived from a target microorganism cell.

When adding ATP hydrolase, a buffer is added to adjust the pH to 6.0 to 8.0, and a protease-free soluble protein and an ATP hydrolase activator are added to lower the activity level of ATP hydrolase. Be optimal. A
TP hydrolase works well when degrading ATP from non-microbial cells. Furthermore, after performing a decomposition treatment of non-microbial ATP with ATP hydrolase, the remaining ATP hydrolase is composed of a chelating agent, vanadate, levamisole, steroid a, chlorhexidine gluconate, and protein hydrolase. Inactivating ATP hydrolase by adding at least one inhibitor selected from the group, and then extracting and quantifying ATP derived from microbial cells by a bioluminescence method is effective for selective detection of ATP derived from microbial cells. there were. Combination of the above series of operations improves detection sensitivity, suppresses variation in measured values,
The present inventors have found that the present invention is effective in improving the reliability, and completed the present invention.

SUMMARY OF THE INVENTION The present invention is a method for measuring the concentration of ATP in a sample containing a mixture of microbial cells and non-microbial cells. A step of taking out the cells, a step of centrifuging the sample to remove a supernatant to form a microbial cell pellet, and adding a buffer to the microbial cell pellet to pH = 6.0.
8.0, a protease-free soluble protein,
Adding an ATP hydrolase and an ATP hydrolase activator, adding an ATP hydrolase inhibitor, adding an ATP extractant to extract ATP from the microbial cells, and releasing ATP from the microbial cells. The present invention relates to a method for measuring the concentration of ATP in microbial cells, which comprises a step of measuring the concentration using a bioluminescence method.

Further, the present invention relates to the above-mentioned measuring method, wherein a chelating agent is mixed in the step of mixing nonionic surfactant to bring out ATP of non-microbial cells outside the cells.

Further, the present invention provides that the ATP hydrolase inhibitor comprises a chelating agent, a vanadate, levamisole, a steroid a having a skeleton represented by the following general formula, chlorhexidine gluconate, and a protein hydrolase. And at least one selected from the group consisting of:

[0013]

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[0014]

BEST MODE FOR CARRYING OUT THE INVENTION

The test method of the present invention is described in detail for a milk sample, which is a sample containing a typical mixture of microbial and non-microbial cells. However, this does not limit the scope of the invention to milk samples. A milk sample to be analyzed is first placed in a centrifuge container, and a chelating agent and a nonionic surfactant are added. The chelating agent added here is added to capture calcium ions in the milk sample and suppress the precipitation of casein.
It is not added to inhibit the activity of TP hydrolase. Therefore, if casein does not interfere with the measurement, it is not necessary to add a chelating agent here. Nonionic surfactants also act as ATP extractants from non-microbial cells.

The centrifuge container containing the mixture is capped and inverted or swirled to mix. The sample is placed in a centrifuge and centrifuged at 10,000 xg (minimum relative centrifugal force) for a minimum of 5 minutes to separate into three layers. The top layer is a white or yellowish cream and milk protein. Below that there is a transparent liquid area which is an intermediate layer,
The bottom layer contains microbial cell pellets. When the other milk components other than the microbial cell pellet are removed by suction, only the microbial cell pellet remains at the bottom of the centrifuge tube. At this time, the chelating agent previously placed is also removed by suction. The size of the microbial cell pellet remaining at the bottom of the tube depends on the number of cells in the milk sample and the type of chelating and nonionic surfactant. Microbial cell pellets also contain other milk components associated with small amounts of cells.

Next, the ATP hydrolase-containing solution is added to the microbial cell pellet remaining at the bottom of the tube and resuspended. The resuspended solution is incubated until the background non-microbial cell-derived or free ATP is hydrolyzed. The ATP hydrolase containing solution contains p
A buffer solution with H = 6.0-8.0, a protease-free soluble protein, and an ATP hydrolase activator are added. The step of adding the ATP hydrolase-containing solution is for decomposing non-microbial cell-derived ATP and free ATP. Most of ATP derived from non-microbial cells has been removed by centrifugation, but the ATP derived from non-microbial cells cannot be completely removed only by centrifugation because the milk sample contains a large amount of somatic cells. . So, AT
These ATPs are hydrolyzed by P hydrolase.
The reason for adjusting the pH to 6.0 to 8.0 is to optimize the activity of the ATP hydrolase. The addition of a protease-free soluble protein causes the ATP hydrolase to act stably. There is. The ATP hydrolase activator is also added in order to allow the enzyme to act stably and suppress the dispersion of measured values.

In the step of adding the solution containing the ATP hydrolase to the microbial cell pellet, it is essential to control the activity of the ATP hydrolase and the reaction conditions as follows. a) The activity value of ATP hydrolase is set to ΔlogRLU / min = 1.0 to 6.0. b) The reaction temperature of ATP hydrolase is set to 20 to 40 ° C. c) The reaction time of ATP hydrolase is 5 minutes or more. In the present invention, the activity value of the ATP hydrolase is expressed as A
It is expressed as the rate at which ATP as a substrate is hydrolyzed by TP hydrolase. Therefore, the measurement of the enzyme activity is carried out by measuring the decrease in the amount of ATP in the test solution over time, and the amount of ATP in the test solution can be measured by using a firefly luciferase which is a sensitive method for quantifying ATP. Relative luminescence obtained from luminescence reaction (RLU; Relative luminescence, Relative Light)
Unit) is measured as an index. In the present invention, a constant amount is sampled from the test solution discontinuously, the ATP amount is measured by a bioluminescence reaction, and the logarithmic value of the relative luminescence amount per minute is reduced from the obtained value (ΔlogRLU / min). ) Is calculated and the value is used as the activity value of ATP hydrolase.

In the step of adding ATP hydrolase to the microbial cell pellet, the following reagents can be added. a) Preservative b) Protein SH group protecting agent

An inhibitor is added to the resuspension in which the ATP hydrolase has been actuated and the incubation has been completed in order to inactivate the enzyme. At this time, the concentration of the inhibitor is selected so as to minimize the influence on the activity of the enzyme luciferase in the following bioluminescence method and to effectively inactivate the ATP hydrolase.

The microbial cell A is added to the resuspension containing the inhibitor.
After adding a TP extractant to release ATP from the microbial cells, a luminescent reagent containing luciferin / luciferase is added to cause bioluminescence, and the amount of ATP is quantified using a luminometer.

The milk sample of the present invention includes raw milk, raw goat milk, raw sheep milk, milk, ultra-high temperature sterilized milk, low-temperature long-time sterilized milk, sterilized goat milk, special milk, partially skim milk, skim milk, and processed milk. Powdered milk, cream, skim milk, liquefied ice cream or ice milk or related products, reconstituted milk, raw material of milk including milk or suspension in a sample or a mixture with coffee or the like, Includes all liquid solutions originating from dairy products, such as milk cultures (enrichment cultures). The microorganism of the present invention means a unicellular prokaryote. Substitution of the term bacteria or non-eukaryotic cells is also within the scope of the present invention. Eukaryotic cells refer to organisms in which genetic material is surrounded by nuclei.

The non-microbial cells of the present invention are cells that are not microorganisms, and are mostly somatic cells. Somatic cells are milk-producing animals, such as cows, and are mainly present in milk and are cells other than germ cells among all cells constituting an organism, such as leukocytes, erythrocytes, and platelets derived from body fluids And cells detached from the breast internal tissue and the like.

ATP (adenosine-5'-triphosphate)
Is a nucleotide consisting of adenine, D-ribose and three phosphate groups, is produced by phosphorylation in respiration, fermentation and photosynthesis, and is important in bioenergy metabolism. All living cells contain ATP.

The nonionic surfactant of the present invention is a surfactant having no group capable of dissociating into ions in an aqueous solution, and acts as an ATP extractant for non-microbial cells.
In particular, it is an agent for extracting ATP from non-microbial cells without affecting microbial cells in order to separate and remove non-microbial cells from a milk sample and to separate and concentrate only microbial cells. Non-microbial cell ATP as a function of the non-microbial cell ATP extractor reduces the microbial cell ATP, or damages the microorganism, or jeopardizes the survival of the microorganism
Extractants are not preferred. Therefore, a non-microbial cell ATP extractant which has a mild protein denaturing action, excellent membrane solubilizing ability, contributes to emulsification and dispersion of coagulated milk components, and further facilitates separation and concentration of microbial cells is preferred.

Nonionic surfactants are roughly classified into three groups: 1) higher alcohols, alkylphenols, fatty acids, etc., in which ethylene glycol is bonded as a hydrophilic group, 2) partial esters of polyhydric alcohols,
Monoglycerides which are higher fatty acid glycerol esters, fatty acid esters of sorbitol, and 3) addition polymerization products of polypropylene glycol and polyethylene glycol. Among the above nonionic surfactants, in the present invention, Triton X-100 which is a polyoxyethylene glycol octylphenyl ether is used.
Alternatively, Nonidet P-40 (NP-40) is preferably used, and Triton X-100 is particularly preferable. In particular, it is particularly preferred to add such a nonionic surfactant to the milk sample together with the chelating agent. Most somatic cells are lysed in the process before centrifugation,
After centrifugation, the effect of ATP from non-microbial cells is reduced. The amount of non-ionic surfactant added depends on Triton
When X-100 is used, 0.1 to milk sample
-5%, preferably 0.25-2.5%.

The chelating agent mixed in the step of mixing the nonionic surfactant of the present invention to bring ATP of non-microbial cells out of the cell includes calcium ion, magnesium ion, cadmium ion, beryllium ion, cobalt ion, Nickel ions, copper ions, divalent metal ions such as lead ions, and all molecules or macromolecules that bind to other metal ions, synthetic or natural organic compounds, proteins that can bind to the above ions, carbohydrates,
Replicated or modified products of lipids or biogenic molecules.

The effect of the chelating agent added before the centrifugation operation is the dissociation of casein micelles into sub-micelles in the milk sample (LC Chaplin, J. Dairy Re.
s., 51, 251-257 (1984)). If the milk sample without chelating agent was centrifuged in the same manner, the supernatant of the milk sample did not clarify, and the micellar milk protein was suspended or associated in the centrifuge tube, and the molecular weight increased. Casein micelles settle and form a giant pellet at the bottom, failing to form a clear microbial cell pellet. Chelating agents bind to calcium ions that contribute to micelle structure (SHCLia, Biochemistry, 11, 1818-1821).
(1972)), dissociating the casein micelles. Therefore, a chelating agent that binds to calcium ions is preferred in the present invention. When casein does not hinder the measurement as described above, it is not necessary to add a chelating agent here.

As the chelating agent, ethylenediaminetetraacetic acid (EDTA, trade name Versene), bis-
(O-aminophenoxy) -ethane-N, N, N ',
N′-tetraacetic acid (BAPTA), ethylene glycol-bis- (β-aminoethyl ether) N, N, N ′, N ′
-Tetraacetic acid (EGTA), nitrilotriacetic acid (triglycine, ammonia triacetate, trilone A), trans-
1,2-diaminocyclohexanetetraacetic acid (CDTA),
Oxycarboxylic acids such as diethylenetriaminopentaacetic acid (DTPA) and citric acid and salts thereof, arginine, hypoxanthin, 4,5-dihydroxybenzene-1,3
-Disulfonic acids, crown ether type compounds and all derivatives and precursors of these molecules can be used.

It is very important that the chelating agents of the present invention do not act on the microbial cells to be detected. Chelating agents determined to be particularly excellent in the present invention are nitrilotriacetic acid and ethylenediaminetetraacetic acid (EDTA).
The above nonionic surfactant and chelating agent can be provided as a solution in which both agents are dissolved when a test kit for a milk sample is prepared.

The microbial cell pellet of the present invention is a microbial cell pellet in which, after centrifugation, microbial cells are mainly settled and separated at the bottom of the centrifuge tube. The size of the microbial cell pellet depends on the cell number of the milk sample and the type of chelating agent and / or nonionic surfactant. The microbial cell pellet may include other milk components associated with small amounts of cells. The microbial cell pellet obtained from a 1.0 ml milk sample is about 10-40 μl and is white or off-white in appearance.

The ATP hydrolase of the present invention refers to ATP
Is a hydrolase that cleaves the terminal phosphate group of ADP or AMP to generate inorganic phosphoric acid. The EC (enzyme number) 3.6.1 group (Enzyme Handbook, supervised by Maruo and Tamiya, Asakura Shoten (1982) ) Etc.) are included. Examples thereof include adenosine triphosphatase, apyrase (mainly extracted and purified from potato), and ATP pyrophosphatase. It is preferable to use apyrase which is easily available as a commercial product.

ATP hydrolase degrades ATP derived from non-microbial cells present in milk, and removes ATP derived from microbial cells.
It is used as a means for detecting TP with higher sensitivity. A chelating agent and a solution containing a non-ionic surfactant are added to the milk sample, and microbial cell pellets are separated and concentrated from the milk sample by centrifugation, but non-microbial cells such as somatic cells are originally produced in large amounts. Non-microbial cell-derived ATP cannot be completely removed by centrifugation and remains in a large amount. Therefore, by adding the solution containing the ATP hydrolase, it is possible to completely remove the background non-microbial cell-derived ATP.

In the step of adding the ATP hydrolase to the microbial cell pellet of the present invention, the pH at which the ATP hydrolase acts most stably is 6.0 to 8.0, preferably p-8.0.
Adjust the pH so that H = 6.5-7.8. pH
The pH is adjusted in the "Basic Experimental Methods for Proteins and Enzymes", which indicates a neutral pH range (edited by Horio and Yamashita: Namedo (1981)) 432-
The buffer described on page 435 can be used. Among them, the following buffers summarized by Good's buffer, namely, ME
S (2-morpholinoethanesulfonic acid) buffer, Tri
s (tris (hydroxymethyl) aminomethane) -based buffer, BES (N, N-bis (2-hydroxyethyl)-
2-aminoethanesulfonic acid) buffer, MOPS (3-
Morpholinopropanesulfonic acid) buffer, HEPES
It is preferable to use a buffer (N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid) or the like.

In the step of adding ATP hydrolase to the microorganism cell pellet of the present invention, a protease-free soluble protein is added for the purpose of stabilizing ATP hydrolase. As for the soluble protein to be added, any protein derived from animals, plants, or microorganisms can be used as long as it does not exhibit the protein hydrolysis activity observed in proteins such as papain, trypsin, and chymotrypsin. It is preferable to use one or more of the following soluble proteins collectively referred to as albumin, that is, one or more proteins selected from ovalbumin, lactalbumin, serum albumin, leucosine, legmeline, and ricin. The amount of the protease-free soluble protein added was 0.10 to 5 per ml of milk sample.
mg, preferably 0.25 to 2.5 mg.
Further, in the step of adding ATP hydrolase to the microbial cell pellet, calcium ions, magnesium ions, and the like may be added as an ATP hydrolase activator. The amount of calcium ion is 0.1 to 50 mM, preferably 1 to 20 mM, per 1 ml of milk sample.
And magnesium ion is 10% of calcium ion
It is 1/10 to 1/10.

In the step of adding the ATP hydrolase, the ATP hydrolysis activity value is ΔlogRLU / min = 1.
0-6.0, preferably Δlog RLU / min = 2.0-
5.0 and a reaction temperature of 20 to 40 ° C, preferably 25
℃ ~ 35 ℃, reaction time 5 minutes or more, preferably 10 minutes ~
Preferably, it is 30 minutes. Under these conditions, the background non-microbial cell-derived ATP is sufficiently degraded, so that the conventional non-microbial cell-derived AT
There is no need to repeat the centrifugation-supernatant removal operation to completely remove P. By this method, the operation of quantifying the number of microbial cells in the microbial cell pellet can be significantly simplified. In addition, under these conditions, the degradation of non-microbial cell-derived ATP, which serves as the measurement background, proceeds sufficiently, and the background value can be reduced and stabilized, and the detection sensitivity of microbial cell-derived ATP can be significantly improved. Can be. In addition, ATP hydrolase, protease-free soluble protein, ATP hydrolase activator, p
It is also possible to mix the H adjusting buffer and add it as the same solution.

In the step of adding the ATP hydrolase to the microbial cell pellet, the addition of a preservative and a protein SH group protecting agent may also be effective in activating the ATP hydrolase. Preservatives are added to minimize the effects of microorganisms that enter during the measurement.
Antibiotics such as salts, penicillin GK salts, ampicillin, chloramphenicol, streptomycin, kanamycin, tetracycline, oxytetracycline,
Examples include azides such as sodium azide, phenol compounds such as cresol, and acids such as lactic acid, citric acid, benzoic acid, propionic acid, dehydroacetic acid, oxybenzoic acid, and sorbic acid. Protein SH group protecting agent is
Dithiothreitol (DTT), mercaptoethanol, ethylenediaminetetraacetic acid (EDTA) and the like can be used to add ATP hydrolase stably, and dithiothreitol (DTT) is particularly preferred.

In the step of adding ATP hydrolase to the microbial cell pellet, saccharides such as glucose, fructose, arabinose, xylose, saccharose, trehalose, lactose, maltose, xylobiose, etc. Mannitol, xylitol, dulcitol, sorbitol,
Sugar alcohols such as ribitol and glucitol, PEG
(Polyethylene glycol) # 400, PEG # 600
Water-soluble polymers such as glycine, serine, proline, glutamic acid, and alanine; amines such as trimethylamine; and polyhydric alcohols such as glycerol.

In the step of adding the ATP hydrolase inhibitor of the present invention, a chelating agent, vanadate, levamisole, steroid a, chlorhexidine gluconate, and protein hydrolyzate are used to inactivate the ATP hydrolase. It is preferable to use at least one inhibitor selected from the group consisting of enzymes.

As a chelating agent used for inactivating ATP hydrolase, ethylenediaminetetraacetic acid (EDTA, trade name Versene), bis- (O-aminophenoxy) -ethane-N, N, N ', N'-tetraacetic acid (BAPTA), ethylene glycol-bis- (β-
Aminoethyl ether) N, N, N ', N'-tetraacetic acid (EGTA), nitrilotriacetic acid (triglycine, ammonia triacetate, trilone A), trans-1,2-diaminocyclohexanetetraacetic acid (CDTA), Oxycarboxylic acids such as diethylenetriaminopentaacetic acid (DTPA) and citric acid and salts thereof, arginine, hypoxanthin, 4,5-dihydroxybenzene-1,3-disulfonic acid, N- (acetamido) iminodiacetic acid (ADA);
Mention may be made of dipicolinic acid, crown ether type compounds and all derivatives and precursors of these molecules.

It is very important that the chelating agents of the present invention do not act on the microbial cells to be detected. Further, a chelating agent that does not affect the bioluminescence method using luciferin / luciferase is preferable. Chelating agents determined to be particularly excellent in the present invention are nitrilotriacetic acid, ethylenediaminetetraacetic acid (EDTA), N- (acetamido) iminodiacetic acid (ADA), and citric acid.

Vanadate, levamisole, steroid a used for inactivation of ATP hydrolase,
Chlorhexidine gluconate is all known as an inhibitor of membrane ATPase (see “Biochemical Handbook” edited by Imura et al., Maruzen (1984), etc.), but the inactivation of the ATP hydrolase used in the present invention. It was also confirmed that it was effective for conversion. Of these, as vanadate,
Examples include ammonium metavanadate, sodium metavanadate, and calcium metavanadate. In addition, examples of levamisole include levamisole and levamisole hydrochloride.

The steroid a used for inactivating ATP hydrolase is a steroid having a skeleton (card-20 (22) -enolide) represented by the following general formula:
Specific examples include digitoxin, gitoxin, G-strophantin, K-strophantoside and the like.

[0044]

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As a protein hydrolase used for inactivating ATP hydrolase, EC (enzyme number) 3.4 group (see Enzyme Handbook, supervised by Maruo and Tamiya, Asakura Shoten (1982), etc.) Although a hydrolase acting on a peptide bond classified into the following can be used, chymotrypsin, trypsin, papain, ficin, bromelain and the like are particularly preferable.

The concentration of the inhibitor used in the step of adding the ATP hydrolase inhibitor of the present invention is a concentration that does not affect the luciferase enzyme activity and effectively inactivates the ATP hydrolase. It is desirable. Specifically, it is desirable that each inhibitor has the following concentration. Chelating agent; 0.01-20 mM, preferably 0.1-
10 mM. Vanadate; 0.01-5 mM, preferably 0.1-
2 mM. Levamisole; 0.01-20 mM, preferably 0.1
〜１０10 mM. Steroid a; 0.1-20 mM, preferably 2-10
mM. Chlorhexidine gluconate; 0.01-1%, preferably 0.05-0.5% Protein hydrolase; 0.1-5 mg / ml, preferably 0.5-2 mg / ml. In addition, two or more kinds of inhibitors may be used as a mixture.

The ATP extract of the present invention is an agent used for the purpose of acting on microbial cells separated and concentrated from a milk sample and releasing ATP present in the microbial cells outside the microbial cells. A lysing agent for microbial cells that alters or destroys its structure, comprising a surfactant,
It includes any cell membrane solubilizing agent of natural or synthetic origin, such as acids, alkalis, enzymes, salts, chelating agents, organic solvents, and the like. The ATP extractant may contain the above ATP hydrolase inhibitor, and may be in the same solution.

A substance which significantly diminishes or quenches the bioluminescence emitted as a result of a subsequent ATP / luciferin / luciferase reaction as an action of the ATP extractant of the present invention, or an agent having such conditions, such as zinc ion , Cations such as calcium ions, or chloride ions, iodine ions,
Anions such as phosphate ions, or mercury, chromium,
Chemicals containing heavy metals such as copper and lead, or extremely acidic
Drugs having alkaline conditions are not preferred. Conventionally used trichloroacetic acid is strongly acidic and is not a very good ATP extractant. Accordingly, an ATP extractant that has a mild denaturing effect on luciferase, has excellent membrane solubilizing ability, and does not significantly change the detection of luminescence is preferable. As the ATP extractant, cationic surfactants belonging to quaternary ammonium salts such as cetyltrimethylammonium bromide (CTAB), dodecyltrimethylammonium bromide, cetylpyridinium chloride, benzalkonium chloride, benzethonium chloride, dodecyl sulfate Anionic surfactants such as sodium, Tri
ton X-100, Nonidet P-40 (NP
-40). Among them, the quaternary ammonium salt is preferable because it can be rapidly extracted and has little inhibition on the luciferase reaction.

In the present invention, a method for quantifying ATP using firefly luciferase (MA DeLuca, Advances in Enzymol)
ogy, 44, 37-68 (1976)) is used to quantify the number of microbial cells in milk samples. This ATP quantification method can be applied to quantification of either eukaryotic or prokaryotic cell numbers. The quantification of the number of microbial cells present in the sample is calculated using the relationship between the measured amount of ATP in the sample and the average amount of ATP present in the microbial cells.

Since the firefly luciferase used as a means for detecting ATP is unstable with respect to temperature, the room temperature at the time of measurement reflects the amount of luminescence, that is, the measured value, and the amount of ATP present in the test solution is the same. The measured value varies in spite of. That is, when the same amount of ATP is measured, the light emission amount is maximum around 20 ° C., and the light emission amount decreases before and after that. Therefore, in order to maximize the detection sensitivity, reduce the variation in the measurement results, and increase the reliability, the temperature of the firefly luciferase reaction and the room temperature during the measurement are set to 30 ° C. or lower, preferably 15 to 25 ° C.

[0051]

Hereinafter, the present invention will be described with reference to Examples.
The scope of the present invention is not limited only to these examples.

(Example 1) 1) Neutrient Broth (NB) 30 ml in 5 ml of liquid medium
The following five types of microorganisms, which have been cultured with shaking at 0 ° C. overnight, are serially diluted with sterile water every 10-fold. Serattia liquefaciens PB 1707 Pseudomonas aeruginosa ATCC 9027 Enterobacter spp. Bacillus subtilis ATCC 6633 Staphylococcus aureus ATCC 6538 2) To a 1.5 ml centrifuge tube with a lid, add 1 ml of the following milk sample. Milk sample; commercially available ultra-high temperature pasteurized milk 3) Take 10 μl from each concentration of diluent of 1) and 2)
And incubate at room temperature for about 10 minutes. 4) Add 500 μl of an aqueous solution containing a chelating agent and a nonionic surfactant shown below, and close the centrifuge tube. Composition with water containing chelating agent and nonionic surfactant; polystyrene latex 0.01% Triton X-100 0.5% EDTA 0.15M

5) After mixing, centrifugation is performed at 12,000 × g for 5 minutes to separate the microorganism cell pellet from other milk components. 6) The milk component other than the pellet is removed by suction from the separated microbial cell pellet with an aspirator. 7) Add the following ATP hydrolase-containing aqueous solution 1 to the microbial cell pellet remaining at the bottom of the centrifuge tube.
Add 00 μl, stir and suspend, incubate at 25 ° C. for 10 minutes to degrade non-microbial ATP. ATP hydrolase-containing aqueous solution composition: Apyrase (derived from potato, Grade manufactured by Sigma) Enzyme activity (ΔlogRLU) = 5.0 Serum albumin (derived from bovine, manufactured by INTERGEN) 1 mg / ml CaCl _{2 2} mM MgCl ₂ 0.05 mM NaN ₃ 0.1% EDTA 0.1 mM DTT 1 mM MOPS / NaOH buffer (pH 7.0) 25 mM 8) After the reaction, 100 μl of an aqueous solution containing an ATP hydrolase inhibitor shown below was added, and the mixture was stirred and suspended to remove apyrase. Activate. ATP hydrolase inhibitor-containing aqueous solution composition; EDTA 0.2 mM 9) After the reaction, the suspension was transferred to a measurement cell, and 100 μl of the following ATP extractant was added and reacted for 10 seconds. Release. ATP extractant composition; CTAB 0.0075% 10) ATP released from microorganisms was converted to luciferin /
100 μl of a luminescent reagent containing firefly luciferase (manufactured by Toyo Ink Mfg. Co., Ltd., trade name: Bushiro) is added,
The integrated value of the luminescence amount for 10 seconds after the addition is measured. Relative luminescence (logRLU) of sample A, which is artificially contaminated milk
And 11) On the other hand, from each concentration of the diluent of 1) as a positive control, 10 μl of the same amount as that added to sample A, that is, the number of bacteria equivalent to the number of bacteria contained in sample A, was transferred to the measurement cell for luminometer. 100 μl of the ATP extractant is collected, lysed for 10 seconds, and 100 μl of a luminescence reagent (trade name: Bacterial Shiro manufactured by Toyo Ink Mfg. Co., Ltd.) is added, and the integrated value of the luminescence amount for 10 seconds is measured. This is compared with the relative luminescence of sample B (logR
LU). 12) Samples A and B have the same number of bacteria. Therefore, after 100 μl of the diluted solution of each concentration of 1) was applied to a standard agar medium, the number of colonies grown in culture at 30 ° C. for 48 hours was counted, and the number of bacteria contained per 10 μl of each diluted solution was calculated. This was used to determine the bacterial counts (log
CFU).

The number of bacteria (logCF) determined for each microorganism
FIGS. 1 and 2 show the relationship between U) and the relative light emission amount (logRLU).
-It is shown in Fig.3, Fig.4 and Fig.5. Sample A (□) and Sample B
In both cases (◇), there is a linearity between the relative luminescence (logRLU) and the relative luminescence (logRLU) when the number of bacteria (logCFU) is in the range of 3 to 8.
U) can be determined. In addition, the relative luminescence (logRL) between sample A, which is artificially contaminated milk, and sample B, which does not contain milk.
A good correlation was observed between U) and the number of bacteria (log CFU), and only ATP from microbial cells was measured from milk, which is a typical sample containing a mixture of microbial cells and non-microbial cells, with little loss of ATP. And the number of bacteria (log C
In the range of 3 to 8 in FU), the relative luminescence (logRL)
It is shown that the number of bacteria (log CFU) can be quantified from the value of U).

(Example 2) Hereinafter, Examples 2 to 8 and Comparative Example 1 include Staphy, a typical milk sample contaminating microorganism.
The result of having tested lococcus aureus ATCC 6538 is shown. In the aqueous solution containing the ATP hydrolase inhibitor shown in 8) of Example 1, an aqueous solution containing 1.0 mM of ADA was used in place of EDTA used as an inhibitor, and other operations were performed on sample A. Proceed exactly as in Example 1,
The relationship between the number of bacteria (log CFU) and the relative luminescence (log RLU) was determined. FIG. 6 shows the results. In FIG. 6, ○ indicates the result of Example 2, and □ indicates the result of Sample A of Example 1. Also when ADA is used as an inhibitor of ATP hydrolase, there is a linearity between the relative luminescence (logRLU) and the number of bacteria (logCFU) in the range of 3 to 8,
In addition, a good correlation was observed with Sample 1 of Example 1, and the number of bacteria (log CFU) can be quantified from the value of relative luminescence (logRLU) between 3 and 8 in the number of bacteria (log CFU). It is shown.

Example 3 In the aqueous solution containing the ATP hydrolase inhibitor shown in 8) of Example 1, an aqueous solution containing 5.0 mM citric acid was used in place of EDTA used as an inhibitor. Other operations were performed in exactly the same manner as in Example 1 except for the number of bacteria (log CFU) and the relative luminescence (log RL).
U). FIG. 7 shows the results. In FIG. 7, ○
Indicates the results of Example 3, and □ indicates the results of Sample A of Example 1. When citric acid was used as an inhibitor of ATP hydrolase, the relative luminescence (logRLU) was linear with the number of bacteria (log CFU) in the range of 3 to 8, and the sample of Example 1 was also used. A good correlation was also observed between 1 and 1, and the number of bacteria (log CFU) was between 3 and 8,
From the value of relative luminescence (logRLU), the number of bacteria (logCF
U) can be determined.

(Embodiment 4) A shown in 8) of Embodiment 1
In the TP hydrolase inhibitor-containing aqueous solution, an aqueous solution containing 0.5 mM of sodium metavanadate was used in place of EDTA used as the inhibitor, and the other operations were performed in exactly the same manner as in Example 1, except for the sample A. , The relationship between the number of bacteria (log CFU) and the relative luminescence (log RLU) was determined. FIG. 8 shows the results. In FIG. 8, ○ indicates Example 4.
□ shows the results of sample A of Example 1. Also when sodium metavanadate was used as an inhibitor of ATP hydrolase, the relative luminescence (logRLU) was linear with the number of bacteria (log CFU) in the range of 3 to 8, and also in Example 1. A good correlation was also observed between Sample 1 and the number of bacteria (log CFU).
gCFU) can be determined.

(Example 5) A shown in 8) of Example 1
In the TP hydrolase inhibitor-containing aqueous solution, an aqueous solution containing 1.0 mM of levamisole hydrochloride was used in place of EDTA used as the inhibitor, and the other operations were performed in exactly the same manner as in Example 1 except for the sample A. , The number of bacteria (lo
gCFU) and the relative luminescence (logRLU) were determined. FIG. 9 shows the results. In FIG. 9, ○ indicates the results of Example 5, and □ indicates the results of Sample A of Example 1.
When levamisole hydrochloride was used as an ATP hydrolase inhibitor, the relative luminescence (logRLU) was linear with the number of bacteria (log CFU) in the range of 3 to 8, and A good correlation was also observed between Sample 1 and the number of bacteria (log CFU). The value of the relative luminescence (log RLU) was between 3 and 8 to determine the number of bacteria (log CFU).
Is quantified.

(Embodiment 6) A shown in 8) of Embodiment 1
In the TP hydrolase inhibitor-containing aqueous solution, an aqueous solution containing 4.0 mM of G-strophantin was used instead of EDTA used as the inhibitor, and the other operations were performed in exactly the same manner as in Example 1 except for the sample A. Go to the bacteria count (lo
gCFU) and the relative luminescence (logRLU) were determined. The results are shown in FIG. In FIG. 10, .largecircle. Indicates the result of Example 6, and .quadrature. Indicates the result of Sample A of Example 1. Also when G-strophantin was used as an inhibitor of ATP hydrolase, there was linearity between the relative luminescence (logRLU) when the number of bacteria (logCFU) was in the range of 3 to 8, and A good correlation was also observed between Sample No. 1 and Sample No. 1, and the number of bacteria (log CFU) was between 3 and 8,
From the value of relative luminescence (logRLU), the number of bacteria (logCF
U) can be determined.

(Example 7) A shown in 8) of Example 1
In the TP hydrolase inhibitor-containing aqueous solution, an aqueous solution containing 0.1% of chlorhexidine gluconate was used in place of EDTA used as the inhibitor, and the other operations were performed in exactly the same manner as in Example 1 except for the sample A. Thus, the relationship between the number of bacteria (log CFU) and the relative luminescence (log RLU) was determined. The results are shown in FIG. In FIG. 11, .largecircle. Indicates the result of Example 7, and .quadrature. Indicates the result of Sample A of Example 1. When chlorhexidine gluconate was used as an ATP hydrolase inhibitor, the bacterial count (logCF
When U) is in the range of 3 to 8, there is a linear relationship with the relative luminescence (log RLU), and a good correlation is also observed with the sample 1 of Example 1, and the number of bacteria (log CFU) is It shows that the number of bacteria (log CFU) can be quantified from the value of relative luminescence (log RLU) between 3 and 8.

(Embodiment 8) A shown in 8) of Embodiment 1
In an aqueous solution containing a TP hydrolase inhibitor, chymotrypsin was used instead of EDTA used as an inhibitor.
Other operations were performed in exactly the same manner as in Example 1 using an aqueous solution containing
gCFU) and the relative luminescence (logRLU) were determined. The result is shown in FIG. In FIG. 12, ○ indicates the result of Example 8, and □ indicates the result of Sample A of Example 1. Also when chymotrypsin was used as an inhibitor of ATP hydrolase, the relative luminescence (logRLU) was linear with the number of bacteria (log CFU) in the range of 3 to 8, and the sample 1 of Example 1 A good correlation was also recognized between the number of bacteria (log CFU) and the value of the relative luminescence (log RLU) between 3 and 8 and the number of bacteria (log CFU).
Is quantified.

(Comparative Example 1) A shown in 8) of Example 1
Distilled water was used in place of the TP hydrolase inhibitor-containing aqueous solution, and the other operations were performed on the sample A in exactly the same manner as in Example 1, and the number of bacteria (log CFU) and the relative luminescence (log RLU) were compared. Seeking a relationship. FIG. 13 shows the results. In FIG. 13, ○ indicates the result of Comparative Example 1, and □ indicates the result of Sample A of Example 1. When an ATP hydrolase inhibitor is not used, ATP released from the target microbial cell is decomposed by the coexisting ATP hydrolase, and particularly in a region where the number of bacteria is small, luciferin /
The relative luminescence (logRLU) as measured by luciferase bioluminescence decreases. Therefore, the bacterial count (log
(CFU) and the relative luminescence (logRLU) were not linear, and no good correlation was observed with Sample 1 of the example.

According to the present invention, the time required for the method for selectively measuring ATP derived from microorganisms in a sample containing a mixture of microbial cells and non-microbial cells is reduced, and the operability and detection sensitivity are greatly improved. Non-microbial ATP by nonionic surfactant
Is extracted and decomposed with ATP hydrolase, the ATP hydrolase is inactivated, and the ATP extractant is used to remove microorganism-derived ATP.
In a series of measurement methods of extracting and quantifying by a bioluminescence method, ATP hydrolase is inactivated by a simple operation, so that ATP derived from non-microbial cells can be reduced in a short time without damaging ATP derived from microorganisms. Degradation and dimming of bioluminescence could be minimized. With the combined use of the centrifugation operation, the detection sensitivity of microorganisms in milk samples is 10 ² to 10 ³
CFU / ml. In the conventional method involving centrifugation, it was necessary to repeat centrifugation to remove ATP derived from non-microbial cells. However, in the present invention, good results can be obtained by one centrifugation, and the measurement time is reduced. did it.

[Brief description of the drawings]

FIG. 1 is a diagram showing the relationship between the number of bacteria (log CFU) and the relative luminescence (log RLU) obtained when Serattia liquefacience PB 1707 is used as a microorganism sample.

FIG. 2 is a diagram showing the relationship between the number of bacteria (log CFU) and the relative luminescence (log RLU) obtained when Pseudomonas aeruginosa ATCC 9027 is used as a microorganism sample.

FIG. 3 shows the number of bacteria (log CFU) and relative luminescence (log R) obtained when Enterobacter spp. Is used as a microorganism sample.
LU)

FIG. 4 is a graph showing the relationship between the number of bacteria (log CFU) and the relative luminescence (log RLU) obtained when Bacillus subtilis ATCC 6633 is used as a microorganism sample.

FIG. 5 is a diagram showing the relationship between the number of bacteria (log CFU) and the relative luminescence (log RLU) obtained when Staphylococcus aureus ATCC 6538 is used as a microorganism sample.

FIG. 6 is a diagram showing the relationship between the number of bacteria (log CFU) and the relative luminescence (log RLU) obtained when Staphylococcus aureus ATCC 6538 is used as a microorganism sample.

FIG. 7 is a graph showing the relationship between the number of bacteria (log CFU) and the relative luminescence (log RLU) obtained when Staphylococcus aureus ATCC 6538 is used as a microorganism sample.

FIG. 8 is a graph showing the relationship between the number of bacteria (log CFU) and the relative luminescence (log RLU) obtained when Staphylococcus aureus ATCC 6538 is used as a microorganism sample.

FIG. 9 is a graph showing the relationship between the number of bacteria (log CFU) and the relative luminescence (log RLU) obtained when Staphylococcus aureus ATCC 6538 is used as a microorganism sample.

FIG. 10 is a diagram showing the relationship between the number of bacteria (log CFU) and the relative luminescence (log RLU) obtained when Staphylococcus aureus ATCC 6538 is used as a microorganism sample.

FIG. 11 is a diagram showing the relationship between the number of bacteria (log CFU) and the relative luminescence (log RLU) obtained when Staphylococcus aureus ATCC 6538 is used as a microorganism sample.

FIG. 12 is a diagram showing the relationship between the number of bacteria (log CFU) and the relative luminescence (log RLU) obtained when Staphylococcus aureus ATCC 6538 is used as a microorganism sample.

FIG. 13 is a diagram showing the relationship between the number of bacteria (log CFU) and the relative luminescence (log RLU) obtained when Staphylococcus aureus ATCC 6538 is used as a microorganism sample.

Claims (3)

[Claims] 1. A method for measuring the ATP concentration in a sample containing a mixture of microbial cells and non-microbial cells, comprising the steps of: mixing a nonionic surfactant to bring out ATP of the non-microbial cells outside the cell; Removing the supernatant by centrifugation to form a microbial cell pellet, adding a buffer to the microbial cell pellet to pH = 6.0-8.0, and dissolving protease-free soluble protein, ATP hydrolase and A
Adding a TP hydrolase activator, adding an ATP hydrolase inhibitor, adding an ATP extractant to extract ATP from the microbial cells, and measuring the concentration of ATP released from the microbial cells by a bioluminescence method. A method for measuring the concentration of ATP in a microbial cell, comprising the step of measuring using ATP. 2. The method according to claim 1, wherein a chelating agent is mixed in the step of mixing the nonionic surfactant to bring ATP of the non-microbial cells out of the cells. 3. The inhibitor of ATP hydrolase is selected from the group consisting of a chelating agent, vanadate, levamisole, steroid a having a skeleton represented by the following general formula, chlorhexidine gluconate, and protein hydrolase. 2. The method according to claim 1, wherein the at least one type is used.
Or the measuring method according to claim 2. Embedded image