WO2014092050A1 - Method for measuring cell-derived adenosine triphosphate - Google Patents
Method for measuring cell-derived adenosine triphosphate Download PDFInfo
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- WO2014092050A1 WO2014092050A1 PCT/JP2013/082972 JP2013082972W WO2014092050A1 WO 2014092050 A1 WO2014092050 A1 WO 2014092050A1 JP 2013082972 W JP2013082972 W JP 2013082972W WO 2014092050 A1 WO2014092050 A1 WO 2014092050A1
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- adenosine triphosphate
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y306/00—Hydrolases acting on acid anhydrides (3.6)
- C12Y306/01—Hydrolases acting on acid anhydrides (3.6) in phosphorus-containing anhydrides (3.6.1)
- C12Y306/01005—Apyrase (3.6.1.5), i.e. ATP diphosphohydrolase
Definitions
- the present invention relates to a method for measuring cell-derived adenosine triphosphate, and more specifically, after removing free adenosine triphosphate in a sample using a microcapsule containing adenosine triphosphate degrading enzyme,
- the present invention relates to a method for measuring cell-derived adenosine triphosphate, which comprises detecting cell-derived adenosine triphosphate present in a sample.
- a method of measuring microbial contamination in beverages culture for 2 to 3 days using an agar medium, filter the sample using an agar plate method for measuring the number of colonies produced, or a membrane filter, and attach the filter to the agar medium.
- a membrane filter method of culturing on a filter is generally used. The above method takes time to obtain a measurement result, and lacks rapidity as a process control method.
- a bioluminescence method using luciferase is used as a rapid detection method of adenosine triphosphate (hereinafter referred to as ATP) derived from microbial cells.
- ATP adenosine triphosphate
- This method is based on the principle that the amount of luminescence generated by the oxidation of luciferin is proportional to the amount of ATP in the presence of luciferin, luciferase, and ATP.
- ATP is required to be measured accurately at a concentration of several nanomoles / L.
- Patent Document 1 The first is a method in which a nonionic surfactant is allowed to act on a sample to extract non-microbial cell-derived ATP, followed by filtration to extract concentrated microbial cell ATP and estimating the amount of ATP by bioluminescence. This method requires time for filtration and is complicated.
- non-ionic surfactant is allowed to act, non-microbe-derived ATP is extracted, the supernatant is removed by centrifugation, microbial cell-derived ATP is extracted from a precipitate containing microbial cells, and bioluminescence is used. It is a measurement method. In this method, separation of microorganisms is ensured by repeating the centrifugation operation, but it takes a considerable amount of time, and the recovery of microorganism cells becomes worse as the number of operations increases.
- a non-ionic surfactant or the like is allowed to act on the measurement sample to extract non-microbe-derived ATP, which is then decomposed by an ATP hydrolase such as apyrase, and after the decomposition is completed, the apyrase is fixed with glass beads.
- an ATP hydrolase such as apyrase
- this method when removal or inactivation of apyrase is incomplete, there is a risk that ATP derived from microbial cells is degraded by residual apyrase. As a result, the variation in the ATP value increases, and only about 10 6 CFU / ml can be expected.
- the present invention removes ATP derived from raw materials in a simple operation and in a short time, thereby increasing the contamination of microorganisms.
- An object of the present invention is to provide a technique for sensitive and accurate determination.
- microcapsule containing ATP hydrolyzing enzyme meets the above-mentioned purpose.
- ATP hydrolase is encapsulated in a semi-permeable microcapsule, and ATP that has entered the capsule through the microcapsule thin film is decomposed by an enzymatic reaction.
- the microcapsules are removed, the ATP derived from the microorganism is extracted, and the amount thereof is detected by a bioluminescence reaction. It was removed in time, and it was found that the contamination of microorganisms could be determined with high sensitivity and accuracy, and the present invention was completed (FIG. 1).
- the method of the present invention can be used not only for microorganisms but also for measurement of cells such as animal cells and plant cells.
- the gist of the present invention is as follows. (1) Using a microcapsule encapsulating adenosine triphosphate degrading enzyme, after decomposing free adenosine triphosphate in the sample, adenosine triphosphate is extracted from cells present in the sample not containing the microcapsule. A method for measuring adenosine triphosphate derived from a cell, comprising measuring the extracted adenosine triphosphate.
- the adenosine triphosphate degrading enzyme is at least one enzyme selected from the group consisting of apyrase, acid phosphatase, alkaline phosphatase, hexokinase, nuclease P and deaminase.
- the film of the microcapsule is a semipermeable membrane that allows permeation of ions having a molecular weight of 1000 or less and does not permeate adenosine triphosphate degrading enzyme.
- the coating of the microcapsule includes at least one material selected from the group consisting of alginic acid, methoxy pectin, cellulose sulfate, gelatin, chitosan, carrageenan and wax, according to any one of (1) to (3) the method of.
- a reagent for measuring adenosine triphosphate comprising a microcapsule encapsulating adenosine triphosphate degrading enzyme.
- An adenosine triphosphate measurement kit comprising the adenosine triphosphate measurement reagent according to (7).
- ATP derived from a raw material can be removed with a simple operation in a short time, and contamination of microorganisms can be determined with high sensitivity and accuracy.
- This specification includes the contents described in the specification and / or drawings of Japanese Patent Application No. 2012-270933, which is the basis of the priority of the present application.
- the present invention uses a microcapsule encapsulating adenosine triphosphate degrading enzyme to decompose free adenosine triphosphate in a sample, and then extracts adenosine triphosphate from cells present in the sample not containing the microcapsule. And providing a method for measuring adenosine triphosphate derived from a cell, comprising measuring the extracted adenosine triphosphate.
- adenosine triphosphate (ATP) degrading enzyme examples include, but are not limited to, apyrase, acid phosphatase, alkaline phosphatase, hexokinase, nuclease P, deaminase and the like.
- Microcapsules are inorganic pigments such as titanium dioxide, zinc white (zinc oxide), iron oxide, chromium oxide, iron black, cobalt blue, alumina white, iron oxide yellow, viridian, zinc sulfide, lithopone, cadmium yellow, vermilion, cadmium.
- inorganic pigments such as titanium dioxide, zinc white (zinc oxide), iron oxide, chromium oxide, iron black, cobalt blue, alumina white, iron oxide yellow, viridian, zinc sulfide, lithopone, cadmium yellow, vermilion, cadmium.
- the microcapsule film only needs to contain ATP-degrading enzyme and have semi-permeability to allow ATP to pass through.
- ions such as ATP, ADP, AMP, pyrophosphate and the like having ions with a molecular weight of 1000 or less
- a semipermeable membrane that permeates and does not permeate ATP-degrading enzyme is suitable.
- the average particle size (d 50 ) of the microcapsules is suitably 10 ⁇ m to 1 mm, preferably 50 ⁇ m to 500 ⁇ m, more preferably 100 ⁇ m to 300 ⁇ m.
- the value of standard deviation (particle size distribution), which is an index representing the particle size distribution, is suitably 1% to 300%, preferably 1% to 100%, more preferably 1% to 30%.
- the average particle size and the particle size distribution can be determined by actually measuring the diameter of the capsule with a microscope or the like under a microscope.
- the specific gravity of the microcapsules is suitably 1.0 or more, preferably 1.0 to 3.0, more preferably 1.0 to 2.0. Specific gravity can be measured according to the pipette method (JIS Z8820-2: 2004).
- the content of ATP-degrading enzyme in one capsule is preferably 0.1 ng to 1 mg, preferably 1 ng to 500 ⁇ g, more preferably 1 ng to 1 ⁇ g.
- the amount of ATP-degrading enzyme to be encapsulated in the capsule can be adjusted by the amount of enzyme introduced into the solution before capsule formation.
- the microcapsule film may contain materials such as alginic acid, methoxy pectin, cellulose sulfate, gelatin, chitosan, carrageenan, and wax.
- Samples include tea-based beverages, fruit liquor, juice (fruit juice beverages, plant-based beverages), cider, cola, carbonated water, syrup, coffee beverages (including milk), tea beverages (including milk), and nourishing beverages , Mineral water, beer, sake refined sake, refined sake sake, grilled sake, synthetic refined sake, whiskey, miso (including straightening), gin, vodka, brandy, condiment liquor, liqueur, rum, old liquor, plum wine, marine products
- processed foods such as processed meat products, processed milk products, processed vegetable products, processed fruit products, oil and fat foods, taste foods, seasonings, confectionery, frozen foods, retort foods, canned foods, bottled foods, and instant foods
- processed foods such as processed meat products, processed milk products, processed vegetable products, processed fruit products, oil and fat foods, taste foods, seasonings, confectionery, frozen foods, retort foods, canned foods, bottled foods, and instant foods
- the reaction is preferably carried out at a temperature of 15 to 45 ° C. under a pH of 4.0 to 10.0 for 1 to 60 minutes.
- the reaction between ATP and ATP-degrading enzyme is preferably carried out in a reaction system in which the concentration of ATP-degrading enzyme is 1 ng / ml to 100 mlmg / ml.
- the amount of ATP present in the reaction system may be an amount that can be completely decomposed by the ATP-degrading enzyme present in the reaction system.
- an appropriate diluent for example, Tris buffer, HEPES buffer
- Solution MES buffer solution, TES buffer solution, pure water, etc.
- a suitable amount of the diluted solution may be reacted with ATP-degrading enzyme.
- the sample may be in a state that does not include microcapsules.
- the sample not containing microcapsules include a sample obtained by separating the microcapsule by an operation such as filtration and centrifugation, and a supernatant obtained by precipitating the microcapsule in the sample by natural precipitation or coagulation precipitation.
- the present invention is not limited to this. Or you may isolate
- a charged bead can be obtained by immersing the completed alginate beads (microcapsules) in a solution containing an amino group or a carboxyl group.
- the solution containing an amino group include a solution containing a substance such as methylamine, piperidine, spermine, spermidine, aniline, pyridine, triethanolamine and the like.
- solutions containing carboxyl groups include unsaturated carboxylic acids (linoleic acid, oleic acid, etc.), hydroxy acids (malic acid, citric acid, etc.), aromatic carboxylic acids (phthalic acid, benzoic acid, etc.), dicarboxylic acids (malonic acid) And a solution containing a substance such as succinic acid).
- the microcapsule and the sample can be separated by collecting the magnetic material in the microcapsule and collecting it by magnetic force.
- the magnetic material include iron oxide, cobalt, ferrite, chromium oxide, and the like, and these may be enclosed inside a bead (microcapsule). That is, a magnetic substance can be enclosed inside a bead by dropping together with a substance that becomes an enzyme, a dye (colorant), and a film.
- a commercially available ATP extraction reagent may be used.
- ATP extracted from cells can be measured by a luciferin / luciferase reaction, but is not limited to this method.
- Detection of ATP by the luciferin / luciferase reaction can be performed by emitting light using a commercially available luciferin / luciferase luminescence reagent (LL luminescence reagent) and measuring the luminescence with a luminometer.
- the luciferin / luciferase reaction is a reaction that depends on the amount of ATP. Luciferin and ATP react to form adenylate luciferin, and this adenylate luciferin and oxygen are decomposed by oxidative decarboxylation in the presence of the luciferase enzyme, and a part of the energy obtained in the process of this reaction is luminescence. appear.
- ATP By quantifying this luminescence, ATP can be quantified.
- concentration of luciferase in the luminescent reaction system luciferin / luciferase 0.1 [mu] g / mL ⁇ 100 [mu] g / mL are suitable, preferably from 1 ⁇ g / mL ⁇ 20 ⁇ g / mL.
- Luciferase is a firefly luciferin derived from a beetle, that is, a multi-heterocyclic organic acid D-( ⁇ )-2- (6′hydroxy-2′-benzothiazolyl) - ⁇ 2-thiazoline-4-carboxylic acid (hereinafter specifically described) Unless otherwise indicated, it is expressed as “luciferin”). It is an enzyme that catalyzes oxidation and emits photons. It is an enzyme that emits photons. Contains all enzymes. This includes enzymes in which the stability and luminescence properties of the enzyme protein itself have been artificially altered by recombinant DNA technology or mutation technology.
- the concentration of luciferin is suitably 0.001 mM to 100 mM, preferably 0.01 mM to 10 mM.
- Luciferin is a beetle-derived firefly luciferin (Coleoptera luciferin), that is, a multi-heterocyclic organic acid D-( ⁇ )-2- (6′hydroxy-2′-benzothiazolyl) - ⁇ 2-thiazoline-4-carboxylic acid) Often this includes those extracted and purified directly from beetles and those that are chemically synthesized.
- a luminescent substrate which is a derivative of beetle luciferin and has luminescence activity after digestion with an enzyme.
- Such beetle luciferin derivatives include 4-methyl-D-luciferin, D-luciferyl-L-methionine, 6-O-galactopyranosyl-luciferin, DEVD-luciferin, luciferin-6'methyl ester, luciferin 6 Examples include '-chloroethyl ester, 6'-deoxyluciferin, and luciferin 6'benzyl ester.
- Luciferin and its derivatives may be in the form of a salt. Examples of the salt include potassium salt and sodium salt.
- quantification of cell-derived ATP quantification of cells (quantity of living cells, if known cells can be quantified with high sensitivity and accuracy, and unknown cells can also be detected), etc.
- the cell may be any cell such as a microbial cell, an animal cell, or a plant cell.
- the method of the present invention can be used for detection and quantification of microorganisms required for food hygiene management and the like, and inspection of the degree of cleaning. For example, by using the luciferin / luciferase reaction, 10 -16 moles of ATP can be detected, and high-sensitivity detection such as about 10 3 in bacteria and several tens in yeast and fungi is possible. Furthermore, it is possible to quickly measure that the time required from the ATP removal to the light emission amount measurement / judgment is within one hour.
- the present invention also provides a microcapsule that encapsulates ATP-degrading enzyme.
- Microcapsules are inorganic pigments such as titanium dioxide, zinc white (zinc oxide), iron oxide, chromium oxide, iron black, cobalt blue, alumina white, iron oxide yellow, viridian, zinc sulfide, lithopone, cadmium yellow, vermilion, cadmium.
- inorganic pigments such as titanium dioxide, zinc white (zinc oxide), iron oxide, chromium oxide, iron black, cobalt blue, alumina white, iron oxide yellow, viridian, zinc sulfide, lithopone, cadmium yellow, vermilion, cadmium.
- Red, yellow lead, molybdate orange, zinc chromate, strontium chromate, agate white carbon, clay, talc, ultramarine, precipitated barium sulfate, barite powder, calcium carbonate, lead white ferrocyanide (bituminous), phosphate (manganese violet) , Carbon (carbon black), organic pigments, rhodamine lake, methyl violet lake, quinoline yellow lake, malachite green lake, alizarin lake, carmine 6B, lakeette C, disazo yellow, lakelet 4R, chromophthalo yellow 3G, black Phthascarlet RN, Nickel Azo Yellow, Permanent Orange HL, Phthalocyanine Blue, Phthalocyanine Green, Flavanthrone Yellow, Thioindigo Bordeaux, Perinone Red, Dioxadone Violet, Quinacridone Red, Naphthol Yellow S, Pigrant Green B, Lumogen Yellow, Signal Red, Bovine serum albumin, gelatin, trehalose or the like may
- microcapsule The material and physical properties of the microcapsule have been described above.
- Microcapsules containing ATP-degrading enzyme can be any known method such as phase separation, submerged drying, melt dispersion cooling, spray drying, pan coating, interfacial polymerization, in-situ polymerization, submerged curing coating. You may manufacture by the method.
- ATP can be decomposed and removed using microcapsules containing ATP-degrading enzyme.
- ATP derived from the ingredients in the beverage is removed, and then ATP derived from cells such as microorganisms present in the sample is extracted and measured to measure ATP derived from cells in the sample. be able to. Therefore, the present invention provides an ATP measurement reagent comprising a microcapsule that encapsulates an ATP-degrading enzyme.
- the ATP-degrading enzyme-encapsulating capsule may be included in any form such as a lyophilized product or a suspension (capsule suspension).
- a suspension the capsule may be dispersed in an appropriate solvent (for example, Tris buffer, HEPES buffer, MES buffer, TES buffer, pure water, etc.).
- the ATP measurement reagent of the present invention may further contain a pH adjuster, an antioxidant, a preservative, an antifungal agent and the like.
- the ATP measurement reagent of the present invention can be used for ATP measurement, for example, measurement of cell-derived ATP present in a sample (described above). Therefore, the present invention provides an ATP measurement kit containing the above ATP measurement reagent.
- the ATP measurement kit of the present invention further converts a luciferin / luciferase luminescence reagent, a luminescence reagent solution, an ATP extractant, an ATP standard reagent, a calibration curve that associates the luminescence amount with the ATP amount, an instruction manual, and an ATP amount.
- Software etc. may be included.
- Example 1 Method for preparing ATP-erased beads 1.1 ml of apyrase potato-derived Grade III (Sigma Aldridge), 2 g of sodium alginate (Wako Pure Chemical Industries), and 200 ⁇ l of pigment ink (aqueous pigment ink blue for graph pen ink pen) were dissolved in 200 ml of pure water. 2. The lysate was set in a microbead manufacturing apparatus (Nippon Büch Encapsulator B-390). 3. A droplet having a diameter of about 150 ⁇ m was generated on the microbead manufacturing apparatus. 4).
- the droplet was dropped into a 2% calcium chloride solution. 5.
- the microbeads containing apyrase and pigment ink whose surface was gelled were filtered using a 0.2 ⁇ m pore size filter (Nalgen filter unit). 6).
- the filtrate (microcapsule) on the filter was washed with 10 mM HEPES buffer. 7).
- the microcapsules were resuspended in an appropriate amount of 10 mM HEPES buffer. 8).
- 10 ⁇ l of a 10 ⁇ 7 mole / l ATP standard solution Toyo Benet
- the physical property values of the microbeads are as follows. ⁇ Average particle size: 155.2 um ⁇ Particle size distribution: 16.4% (The microscopic observation (magnification 40) measured the capsule diameter to determine the average particle size and particle size distribution. 100 particles were measured in one observation, and this was repeated three times. The average particle size was the average value of 300 particles. The particle size distribution is indicated by its standard deviation.) Specific gravity: 1.6 (Measured according to the pipette method (JIS Z8820-2: 2004).) -Apyrase content in one capsule: 11.6 ng (The amount of liquid contained inside a capsule having a diameter of 150 um is about 1.8 ul, so it is calculated that 11.6 ng of enzyme is contained in the conditions of this experiment.
- the amount of enzyme contained in the capsule can be adjusted by the amount of enzyme added to the solution before capsule formation.In this experiment, 1.1 mg of apyrase was mixed with 200 ml of solution. When mixed, the amount of encapsulated enzyme is estimated to be 110 ng.>
- ATP-erased beads Necessity of washing operation 6. (Method for preparing ATP-erased beads) Microcapsule suspension (indicated as “microcapsule-containing liquid” in Table 1) and I.8. 10 ⁇ l of a 10 ⁇ 7 mole / l ATP standard solution (Toyo Benet Corporation) was mixed with 100 ⁇ l of each of the supernatants, and reacted for 30 minutes. Then, after mixing 100 microliters of LL luminescent reagent (Fujiro ATP luminescent reagent LL100-1 Toyo Beenet) and making it react for 30 seconds, the luminescence was measured with the luminometer (LB9507 Berthold). The integration time for measurement was 10 seconds.
- LL luminescent reagent Flujiro ATP luminescent reagent LL100-1 Toyo Beenet
- microcapsule-containing liquid in Table 1 contains apyrase in the capsule, it has ATP degradation activity.
- Table 2 shows the measurement results of III of ATP degradation in green tea by bead treatment III (specimen: green tea).
- ATP contained in green tea was attempted to be decomposed by the microcapsule method and the conventional method (method according to the procedure of the Mycosis ATP measurement kit (Toyo B-Net)).
- the microcapsule method 200 ⁇ l of microcapsule solution was added to 200 ⁇ l of green tea, and the mixture was allowed to stand at room temperature for the treatment time shown in Table 2. After the elapse of time, 100 ⁇ l of the sample solution was fractionated, and an equal amount of 100 ⁇ l of ATP luminescence reagent (Toyo Benet) was added to measure the amount of luminescence.
- Table 3 shows the results of detecting the presence of 5 ⁇ 10 5 E. coli cells in 1 ml of the sample (green tea) by detecting the change in the amount of ATP.
- microcapsule microbe contamination test 200 ⁇ l of green tea beverage mixed with E. coli was dispensed into a tube, mixed with 200 ⁇ l of 200 ⁇ l of apyrase-containing microcapsule suspension, and allowed to stand at room temperature for 30 minutes. 100 ⁇ l of the supernatant was collected taking care not to contaminate the microcapsules. Next, an equal amount of 100 ⁇ l of ATP extraction reagent (Fujiro Shiro ATP extraction reagent LL100-2, Toyo Benet) was added to the collected solution, and ATP extraction from microorganisms was performed for 10 seconds.
- ATP extraction reagent Frujiro Shiro ATP extraction reagent LL100-2, Toyo Benet
- the conventional method followed the procedure of the Mycosis ATP measurement kit. That is, 200 ⁇ l of green tea beverage mixed with Escherichia coli was collected in a tube, mixed with 200 ⁇ l of ATP removal reagent (Fujiro ATP removal reagent LL100-3), and allowed to stand at room temperature for 30 minutes. Collect 100 ⁇ l of the supernatant and add 100 ⁇ l of an equal amount of ATP extraction reagent (Fujiro ATP extraction reagent LL100-2, Toyo Benet) to the solution, and perform ATP extraction from microorganisms for 10 seconds. It was.
- the present invention can be used for measuring microbial contamination in foods such as beverages.
Abstract
Provided is a technique for removing cell-derived ATP from a raw material by a simple procedure in a short time, and accurately evaluating microbial contamination at high sensitivity, for the efficient quality control and sampling inspection of food products such as drinks, and other products, containing large amounts of ATP. The method for measuring cell-derived adenosine triphosphate comprises degrading the free adenosine triphosphate in a sample using microcapsules that encapsulate an adenosine triphosphate degrading enzyme, then extracting the adenosine triphosphate from cells present in a microcapsule-free sample, and measuring the extracted adenosine triphosphate.
Description
本発明は、細胞由来のアデノシン三リン酸を測定する方法に関し、より詳細には、アデノシン三リン酸分解酵素を内包するマイクロカプセルを用いて、試料中の遊離アデノシン三リン酸を除去した後、試料中に存在する細胞由来のアデノシン三リン酸を検出することを含む、細胞由来のアデノシン三リン酸を測定する方法に関する。
The present invention relates to a method for measuring cell-derived adenosine triphosphate, and more specifically, after removing free adenosine triphosphate in a sample using a microcapsule containing adenosine triphosphate degrading enzyme, The present invention relates to a method for measuring cell-derived adenosine triphosphate, which comprises detecting cell-derived adenosine triphosphate present in a sample.
飲料中の微生物汚染を測定する方法として、寒天培地を用いて2～3日培養し、生じるコロニー数を計測する寒天平板法や、メンブレンフィルターを用いて試料をろ過し、フィルターを寒天培地に貼り付けてフィルター上で培養するメンブランフィルター法が一般的に用いられている。以上の方法は、測定結果を得るために時間がかかり、工程管理法としては迅速性に欠いている。
As a method of measuring microbial contamination in beverages, culture for 2 to 3 days using an agar medium, filter the sample using an agar plate method for measuring the number of colonies produced, or a membrane filter, and attach the filter to the agar medium. In addition, a membrane filter method of culturing on a filter is generally used. The above method takes time to obtain a measurement result, and lacks rapidity as a process control method.
このような問題を解決すべく、ルシフェラーゼを用いた生物発光法が、微生物細胞由来アデノシン三リン酸（以下ATP）の迅速検出法として利用されている。本法は、ルシフェリン、ルシフェラーゼ、ATPの存在下において、ルシフェリンの酸化により生じる発光量がATP量と比例する原理に基づいている。ATPは数ナノモル/Lの濃度で正確に測定することが要求される。さらに、微生物由来のATPを峻別して測定するために、非微生物由来すなわち原料由来のATPを除去する必要がある。従来、非微生物由来のATPを選択的に除去し、微生物由来のATPのみを選択的に高感度に検出する方法に腐心しており、以下の3つの方法が開発されている（特許文献１）。1つ目は、試料に非イオン界面活性剤を作用させ、非微生物細胞由来ATPを抽出後、ろ過して濃縮された微生物細胞ATPを抽出して生物発光によりATP量を見積もる方法である。本法は、ろ過に時間を要し操作も煩雑である。2つ目として、非イオン性界面活性剤を作用させ、非微生物由来ATPを抽出して遠心分離操作で上清を除去して、微生物細胞を含む沈殿より微生物細胞由来ATPを抽出し生物発光による測定法である。本法は、遠心操作を繰り返すことにより微生物の分離は確実になるが、時間を相当要し、操作回数が増えるごとに微生物細胞の回収が悪くなる。3つ目として、測定試料に非イオン性界面活性剤などを作用させ、非微生物由来ATPを抽出した後、アピラーゼなどのATP加水分解酵素によりこれを分解し、分解完了後アピラーゼをガラスビーズで固定化することにより不動態化する方法がある。しかしこの方法ではアピラーゼの除去あるいは失活が不完全であった場合、微生物細胞由来のATPが残存アピラーゼにより分解されてしまう危険がある。また、その結果、ATPの値のバラツキが大きくなり、106CFU/ml程度しか期待できない。
In order to solve such problems, a bioluminescence method using luciferase is used as a rapid detection method of adenosine triphosphate (hereinafter referred to as ATP) derived from microbial cells. This method is based on the principle that the amount of luminescence generated by the oxidation of luciferin is proportional to the amount of ATP in the presence of luciferin, luciferase, and ATP. ATP is required to be measured accurately at a concentration of several nanomoles / L. Furthermore, in order to distinguish and measure microorganism-derived ATP, it is necessary to remove non-microbial-derived ATP, that is, raw material-derived ATP. Conventionally, the method has been dedicated to selectively removing non-microbe-derived ATP and selectively detecting only microorganism-derived ATP with high sensitivity, and the following three methods have been developed (Patent Document 1). The first is a method in which a nonionic surfactant is allowed to act on a sample to extract non-microbial cell-derived ATP, followed by filtration to extract concentrated microbial cell ATP and estimating the amount of ATP by bioluminescence. This method requires time for filtration and is complicated. Second, non-ionic surfactant is allowed to act, non-microbe-derived ATP is extracted, the supernatant is removed by centrifugation, microbial cell-derived ATP is extracted from a precipitate containing microbial cells, and bioluminescence is used. It is a measurement method. In this method, separation of microorganisms is ensured by repeating the centrifugation operation, but it takes a considerable amount of time, and the recovery of microorganism cells becomes worse as the number of operations increases. Third, a non-ionic surfactant or the like is allowed to act on the measurement sample to extract non-microbe-derived ATP, which is then decomposed by an ATP hydrolase such as apyrase, and after the decomposition is completed, the apyrase is fixed with glass beads. There is a method to passivate it. However, in this method, when removal or inactivation of apyrase is incomplete, there is a risk that ATP derived from microbial cells is degraded by residual apyrase. As a result, the variation in the ATP value increases, and only about 10 6 CFU / ml can be expected.
このような事情に鑑み、原料由来のATP量が高すぎる茶系飲料や果汁飲料などに混入している微生物由来ATPの測定を短時間かつ高感度に測定する技術の開発が強く望まれていた。
In view of such circumstances, development of a technique for measuring microorganism-derived ATP contained in tea-based beverages and fruit juice beverages in which the amount of ATP derived from the raw material is too high in a short time and with high sensitivity has been strongly desired. .
本発明は、ATPを多量に含む飲料などの食品、その他の製品の品質管理・抜き取り検査を効率よく行うため、原料由来のATPを簡便な操作でかつ短時間で除去し、微生物の混入を高感度かつ正確に判定するための技術を提供することを目的とする。
In order to efficiently perform quality control and sampling inspection of foods such as beverages containing a large amount of ATP and other products, the present invention removes ATP derived from raw materials in a simple operation and in a short time, thereby increasing the contamination of microorganisms. An object of the present invention is to provide a technique for sensitive and accurate determination.
発明者らは、これらの問題を解決すべく、鋭意努力した結果、ATP加水分解解酵素を包含したマイクロカプセルが上記目的に合致していることを見出した。
As a result of diligent efforts to solve these problems, the inventors have found that a microcapsule containing ATP hydrolyzing enzyme meets the above-mentioned purpose.
すなわち、半透性を有するマイクロカプセルにATP加水分解酵素を封じ込め、マイクロカプセルの薄膜を通じてカプセル内部に入ったATPを酵素反応により分解する。試料中の原料由来のATPが完全に分解されたらマイクロカプセルを除去し、微生物由来のATPを抽出してその量を生物発光反応により検出することにより、試料由来のATPを簡便な操作でかつ短時間で除去し、微生物の混入を高感度かつ正確に判定できることを見出し、本発明を完成させるに至った（図１）。本発明の方法は、微生物に限らず、動物細胞、植物細胞などの細胞の測定にも利用することができる。
That is, ATP hydrolase is encapsulated in a semi-permeable microcapsule, and ATP that has entered the capsule through the microcapsule thin film is decomposed by an enzymatic reaction. When the ATP derived from the raw material in the sample is completely decomposed, the microcapsules are removed, the ATP derived from the microorganism is extracted, and the amount thereof is detected by a bioluminescence reaction. It was removed in time, and it was found that the contamination of microorganisms could be determined with high sensitivity and accuracy, and the present invention was completed (FIG. 1). The method of the present invention can be used not only for microorganisms but also for measurement of cells such as animal cells and plant cells.
本発明の要旨は以下の通りである。
（１）アデノシン三リン酸分解酵素を内包するマイクロカプセルを用いて、試料中の遊離アデノシン三リン酸を分解した後、マイクロカプセルを含まない試料中に存在する細胞からアデノシン三リン酸を抽出し、抽出されたアデノシン三リン酸を測定することを含む、細胞由来のアデノシン三リン酸を測定する方法。
（２）アデノシン三リン酸分解酵素が、アピラーゼ、酸性ホスファターゼ、アルカリホスファターゼ、ヘキソキナーゼ、ヌクレアーゼＰ及びデアミナーゼからなる群より選択される少なくとも１種の酵素である（１）記載の方法。
（３）マイクロカプセルの被膜が、分子量1000以下のイオンは透過させ、アデノシン三リン酸分解酵素は透過しない半透膜である（１）又は（２）記載の方法。
（４）マイクロカプセルの被膜が、アルギン酸、メトキシペクチン、硫酸セルロース、ゼラチン、キトサン、カラギーナン及びワックスからなる群より選択される少なくとも１種の材料を含む（１）～（３）のいずれかに記載の方法。
（５）１カプセル中のアデノシン三リン酸分解酵素含有量が0.1 ng～1 mgである（１）～（４）のいずれかに記載の方法。
（６）ルシフェリン／ルシフェラーゼ反応により、アデノシン三リン酸を測定する（１）～（５）のいずれかに記載の方法。
（７）アデノシン三リン酸分解酵素を内包するマイクロカプセルを含む、アデノシン三リン酸測定試薬。
（８）（７）記載のアデノシン三リン酸測定試薬を含む、アデノシン三リン酸測定キット。 The gist of the present invention is as follows.
(1) Using a microcapsule encapsulating adenosine triphosphate degrading enzyme, after decomposing free adenosine triphosphate in the sample, adenosine triphosphate is extracted from cells present in the sample not containing the microcapsule. A method for measuring adenosine triphosphate derived from a cell, comprising measuring the extracted adenosine triphosphate.
(2) The method according to (1), wherein the adenosine triphosphate degrading enzyme is at least one enzyme selected from the group consisting of apyrase, acid phosphatase, alkaline phosphatase, hexokinase, nuclease P and deaminase.
(3) The method according to (1) or (2), wherein the film of the microcapsule is a semipermeable membrane that allows permeation of ions having a molecular weight of 1000 or less and does not permeate adenosine triphosphate degrading enzyme.
(4) The coating of the microcapsule includes at least one material selected from the group consisting of alginic acid, methoxy pectin, cellulose sulfate, gelatin, chitosan, carrageenan and wax, according to any one of (1) to (3) the method of.
(5) The method according to any one of (1) to (4), wherein the content of adenosine triphosphate degrading enzyme in one capsule is 0.1 ng to 1 mg.
(6) The method according to any one of (1) to (5), wherein adenosine triphosphate is measured by a luciferin / luciferase reaction.
(7) A reagent for measuring adenosine triphosphate comprising a microcapsule encapsulating adenosine triphosphate degrading enzyme.
(8) An adenosine triphosphate measurement kit comprising the adenosine triphosphate measurement reagent according to (7).
（１）アデノシン三リン酸分解酵素を内包するマイクロカプセルを用いて、試料中の遊離アデノシン三リン酸を分解した後、マイクロカプセルを含まない試料中に存在する細胞からアデノシン三リン酸を抽出し、抽出されたアデノシン三リン酸を測定することを含む、細胞由来のアデノシン三リン酸を測定する方法。
（２）アデノシン三リン酸分解酵素が、アピラーゼ、酸性ホスファターゼ、アルカリホスファターゼ、ヘキソキナーゼ、ヌクレアーゼＰ及びデアミナーゼからなる群より選択される少なくとも１種の酵素である（１）記載の方法。
（３）マイクロカプセルの被膜が、分子量1000以下のイオンは透過させ、アデノシン三リン酸分解酵素は透過しない半透膜である（１）又は（２）記載の方法。
（４）マイクロカプセルの被膜が、アルギン酸、メトキシペクチン、硫酸セルロース、ゼラチン、キトサン、カラギーナン及びワックスからなる群より選択される少なくとも１種の材料を含む（１）～（３）のいずれかに記載の方法。
（５）１カプセル中のアデノシン三リン酸分解酵素含有量が0.1 ng～1 mgである（１）～（４）のいずれかに記載の方法。
（６）ルシフェリン／ルシフェラーゼ反応により、アデノシン三リン酸を測定する（１）～（５）のいずれかに記載の方法。
（７）アデノシン三リン酸分解酵素を内包するマイクロカプセルを含む、アデノシン三リン酸測定試薬。
（８）（７）記載のアデノシン三リン酸測定試薬を含む、アデノシン三リン酸測定キット。 The gist of the present invention is as follows.
(1) Using a microcapsule encapsulating adenosine triphosphate degrading enzyme, after decomposing free adenosine triphosphate in the sample, adenosine triphosphate is extracted from cells present in the sample not containing the microcapsule. A method for measuring adenosine triphosphate derived from a cell, comprising measuring the extracted adenosine triphosphate.
(2) The method according to (1), wherein the adenosine triphosphate degrading enzyme is at least one enzyme selected from the group consisting of apyrase, acid phosphatase, alkaline phosphatase, hexokinase, nuclease P and deaminase.
(3) The method according to (1) or (2), wherein the film of the microcapsule is a semipermeable membrane that allows permeation of ions having a molecular weight of 1000 or less and does not permeate adenosine triphosphate degrading enzyme.
(4) The coating of the microcapsule includes at least one material selected from the group consisting of alginic acid, methoxy pectin, cellulose sulfate, gelatin, chitosan, carrageenan and wax, according to any one of (1) to (3) the method of.
(5) The method according to any one of (1) to (4), wherein the content of adenosine triphosphate degrading enzyme in one capsule is 0.1 ng to 1 mg.
(6) The method according to any one of (1) to (5), wherein adenosine triphosphate is measured by a luciferin / luciferase reaction.
(7) A reagent for measuring adenosine triphosphate comprising a microcapsule encapsulating adenosine triphosphate degrading enzyme.
(8) An adenosine triphosphate measurement kit comprising the adenosine triphosphate measurement reagent according to (7).
本発明により、原料由来のATPを簡便な操作でかつ短時間で除去し、微生物の混入を高感度かつ正確に判定することができるようになった。
本明細書は、本願の優先権の基礎である日本国特許出願、特願2012‐270933の明細書および／または図面に記載される内容を包含する。 According to the present invention, ATP derived from a raw material can be removed with a simple operation in a short time, and contamination of microorganisms can be determined with high sensitivity and accuracy.
This specification includes the contents described in the specification and / or drawings of Japanese Patent Application No. 2012-270933, which is the basis of the priority of the present application.
本明細書は、本願の優先権の基礎である日本国特許出願、特願2012‐270933の明細書および／または図面に記載される内容を包含する。 According to the present invention, ATP derived from a raw material can be removed with a simple operation in a short time, and contamination of microorganisms can be determined with high sensitivity and accuracy.
This specification includes the contents described in the specification and / or drawings of Japanese Patent Application No. 2012-270933, which is the basis of the priority of the present application.
以下、本発明の実施の形態についてより詳細に説明する。
Hereinafter, embodiments of the present invention will be described in more detail.
本発明は、アデノシン三リン酸分解酵素を内包するマイクロカプセルを用いて、試料中の遊離アデノシン三リン酸を分解した後、マイクロカプセルを含まない試料中に存在する細胞からアデノシン三リン酸を抽出し、抽出されたアデノシン三リン酸を測定することを含む、細胞由来のアデノシン三リン酸を測定する方法を提供する。
The present invention uses a microcapsule encapsulating adenosine triphosphate degrading enzyme to decompose free adenosine triphosphate in a sample, and then extracts adenosine triphosphate from cells present in the sample not containing the microcapsule. And providing a method for measuring adenosine triphosphate derived from a cell, comprising measuring the extracted adenosine triphosphate.
アデノシン三リン酸(ATP)分解酵素としては、アピラーゼ、酸性ホスファターゼ、アルカリホスファターゼ、ヘキソキナーゼ、ヌクレアーゼＰ、デアミナーゼなどを例示することができるが、これらに限定されることはない。
Examples of the adenosine triphosphate (ATP) degrading enzyme include, but are not limited to, apyrase, acid phosphatase, alkaline phosphatase, hexokinase, nuclease P, deaminase and the like.
マイクロカプセルは、無機顔料として、二酸化チタン、亜鉛華（酸化亜鉛）、酸化鉄、酸化クロム、鉄黒、コバルトブルー、アルミナ白、酸化鉄黄、ビリジアン、硫化亜鉛、リトポン、カドミウムエロー、朱、カドミウムレッド、黄鉛、モリブデートオレンジ、ジンククロメート、ストロンチウムクロメート、 ホワイトカーボン、クレー、タルク、群青、沈降性硫酸バリウム、バライト粉、炭酸カルシウム、鉛白フェロシアン化物（紺青）、燐酸塩（マンガンバイオレット）、炭素（カーボンブラック）、有機顔料として、ローダミンレーキ、メチルバイオレットレーキ、キノリンエローレーキ、マラカイトグリーンレーキ、アリザリンレーキ、カーミン6B、レーキレットC、ジスアゾエロー、レーキレット4R、クロモフタルエロー3G、クロモフタルスカーレットRN 、ニッケルアゾエロー、パーマネントオレンジHL、フタロシアニンブルー、フタロシアニングリーン、フラバンスロンエロー、チオインジゴボルドー、ペリノンレッド、ジオキサドンバイオレット、キナクリドンレッド、ナフトールエローS、ピグロントグリーンB、ルモゲンエロー、シグナルレッド、アルカリブルー、アニリンブラックなどの着色剤、さらに、酵素保護剤として、牛血清アルブミン、ゼラチン、トレハロースなどを内包してもよい。
Microcapsules are inorganic pigments such as titanium dioxide, zinc white (zinc oxide), iron oxide, chromium oxide, iron black, cobalt blue, alumina white, iron oxide yellow, viridian, zinc sulfide, lithopone, cadmium yellow, vermilion, cadmium. Red, yellow lead, molybdate orange, zinc chromate, strontium chromate, agate white carbon, clay, talc, ultramarine, precipitated barium sulfate, barite powder, calcium carbonate, lead white ferrocyanide (bituminous), phosphate (manganese violet) , Carbon (carbon black), organic pigments, rhodamine lake, methyl violet lake, quinoline yellow lake, malachite green lake, alizarin lake, carmine 6B, lakeette C, disazo yellow, lakelet 4R, chromophthalo yellow 3G, black Phthascarlet RN, Nickel Azo Yellow, Permanent Orange HL, Phthalocyanine Blue, Phthalocyanine Green, Flavanthrone Yellow, Thioindigo Bordeaux, Perinone Red, Dioxadone Violet, Quinacridone Red, Naphthol Yellow S, Pigrant Green B, Lumogen Yellow, Signal Red, Colorants such as alkali blue and aniline black, and bovine serum albumin, gelatin, trehalose and the like may be included as an enzyme protecting agent.
マイクロカプセルの被膜は、ATP分解酵素を封じ込め、かつATPを通過させる半透性を有するものであればよく、このような被膜としては、ATP、ADP、AMP、ピロリン酸などの分子量1000以下のイオンは透過させ、ATP分解酵素は透過しない半透膜が適当である。
The microcapsule film only needs to contain ATP-degrading enzyme and have semi-permeability to allow ATP to pass through. As such a film, ions such as ATP, ADP, AMP, pyrophosphate and the like having ions with a molecular weight of 1000 or less A semipermeable membrane that permeates and does not permeate ATP-degrading enzyme is suitable.
マイクロカプセルの平均粒度（d50）は、10μm～1 mmが適当であり、50μm～500μmが好ましく、100μm～300μmがより好ましい。粒度分布を表す指標である標準偏差（粒度分布）の値は、1%～300%が適当であり、1%～100%が好ましく、1%～30%がより好ましい。平均粒度と粒度分布は、顕微鏡観察でカプセルの直径を顕微鏡などで実測することにより求めることができる。
マイクロカプセルの比重は、1.0以上が適当であり、1.0～3.0が好ましく、1.0～2.0がより好ましい。比重は、ピペット法（JIS Z8820-2:2004）に従い、測定することができる。
また、１カプセル中のATP分解酵素含有量は、0.1 ng～1 mgであるとよく、好ましくは1 ng～500μgであり、より好ましくは1 ng～1μgである。カプセルに内包させるATP分解酵素量は、カプセル形成前の溶液に投入する酵素量により調節が可能である。 The average particle size (d 50 ) of the microcapsules is suitably 10 μm to 1 mm, preferably 50 μm to 500 μm, more preferably 100 μm to 300 μm. The value of standard deviation (particle size distribution), which is an index representing the particle size distribution, is suitably 1% to 300%, preferably 1% to 100%, more preferably 1% to 30%. The average particle size and the particle size distribution can be determined by actually measuring the diameter of the capsule with a microscope or the like under a microscope.
The specific gravity of the microcapsules is suitably 1.0 or more, preferably 1.0 to 3.0, more preferably 1.0 to 2.0. Specific gravity can be measured according to the pipette method (JIS Z8820-2: 2004).
The content of ATP-degrading enzyme in one capsule is preferably 0.1 ng to 1 mg, preferably 1 ng to 500 μg, more preferably 1 ng to 1 μg. The amount of ATP-degrading enzyme to be encapsulated in the capsule can be adjusted by the amount of enzyme introduced into the solution before capsule formation.
マイクロカプセルの比重は、1.0以上が適当であり、1.0～3.0が好ましく、1.0～2.0がより好ましい。比重は、ピペット法（JIS Z8820-2:2004）に従い、測定することができる。
また、１カプセル中のATP分解酵素含有量は、0.1 ng～1 mgであるとよく、好ましくは1 ng～500μgであり、より好ましくは1 ng～1μgである。カプセルに内包させるATP分解酵素量は、カプセル形成前の溶液に投入する酵素量により調節が可能である。 The average particle size (d 50 ) of the microcapsules is suitably 10 μm to 1 mm, preferably 50 μm to 500 μm, more preferably 100 μm to 300 μm. The value of standard deviation (particle size distribution), which is an index representing the particle size distribution, is suitably 1% to 300%, preferably 1% to 100%, more preferably 1% to 30%. The average particle size and the particle size distribution can be determined by actually measuring the diameter of the capsule with a microscope or the like under a microscope.
The specific gravity of the microcapsules is suitably 1.0 or more, preferably 1.0 to 3.0, more preferably 1.0 to 2.0. Specific gravity can be measured according to the pipette method (JIS Z8820-2: 2004).
The content of ATP-degrading enzyme in one capsule is preferably 0.1 ng to 1 mg, preferably 1 ng to 500 μg, more preferably 1 ng to 1 μg. The amount of ATP-degrading enzyme to be encapsulated in the capsule can be adjusted by the amount of enzyme introduced into the solution before capsule formation.
マイクロカプセルの被膜は、アルギン酸、メトキシペクチン、硫酸セルロース、ゼラチン、キトサン、カラギーナン、ワックスなどの材料を含むとよい。
The microcapsule film may contain materials such as alginic acid, methoxy pectin, cellulose sulfate, gelatin, chitosan, carrageenan, and wax.
試料としては、茶系飲料、果実酒、ジュース（果汁飲料、植物系飲料）、サイダー、コーラ、炭酸水、シロップ、コーヒー飲料（ミルク入りを含む）・紅茶飲料（ミルク入りを含む）、滋養飲料、ミネラルォーター、ビール、 清酒、清酒かす、焼ちゅう、合成清酒、ウイスキー、味りん（本直しを含む）、ジン、ウォッカ、ブランデー、薬味酒、リキュール、ラム、老酒、梅酒などの飲料、水産練り製品、肉加工品、乳加工品、野菜加工品、果実加工品、油脂食品、嗜好食品、調味料、菓子類、冷凍食品、レトルト食品、缶詰食品、瓶詰め食品、インスタント食品などの食品を例示することができるが、これらに限定されることはない。
Samples include tea-based beverages, fruit liquor, juice (fruit juice beverages, plant-based beverages), cider, cola, carbonated water, syrup, coffee beverages (including milk), tea beverages (including milk), and nourishing beverages , Mineral water, beer, sake refined sake, refined sake sake, grilled sake, synthetic refined sake, whiskey, miso (including straightening), gin, vodka, brandy, condiment liquor, liqueur, rum, old liquor, plum wine, marine products Examples of processed foods such as processed meat products, processed milk products, processed vegetable products, processed fruit products, oil and fat foods, taste foods, seasonings, confectionery, frozen foods, retort foods, canned foods, bottled foods, and instant foods However, it is not limited to these.
ATP分解酵素を内包するマイクロカプセルと試料を混合することにより、試料中の遊離ATPがマイクロカプセル中のATP分解酵素と反応して分解される。反応は、15～45℃の温度で、pH4.0～10.0の条件下で、1～60分間行うとよい。ATPとATP分解酵素の反応は、ATP分解酵素の濃度が1ng/ml～100 mg/mlとなる反応系で行うとよい。反応系に存在するATPの量は、反応系に存在するATP分解酵素で完全に分解する量であればよく、それよりも多い場合には、適当な希釈剤（例えば、トリス緩衝液、HEPES緩衝液、MES緩衝液、TES緩衝液、純水等）で希釈した後、適量の希釈液をATP分解酵素と反応させるとよい。
¡By mixing the microcapsule containing ATP-degrading enzyme and the sample, free ATP in the sample reacts with the ATP-degrading enzyme in the microcapsule and decomposes. The reaction is preferably carried out at a temperature of 15 to 45 ° C. under a pH of 4.0 to 10.0 for 1 to 60 minutes. The reaction between ATP and ATP-degrading enzyme is preferably carried out in a reaction system in which the concentration of ATP-degrading enzyme is 1 ng / ml to 100 mlmg / ml. The amount of ATP present in the reaction system may be an amount that can be completely decomposed by the ATP-degrading enzyme present in the reaction system. If it is more than that, an appropriate diluent (for example, Tris buffer, HEPES buffer) is used. Solution, MES buffer solution, TES buffer solution, pure water, etc.) and then a suitable amount of the diluted solution may be reacted with ATP-degrading enzyme.
反応後、試料中に存在する細胞からATPを抽出し、抽出されたATPを検出する。試料は、マイクロカプセルを含まない状態であるとよい。マイクロカプセルを含まない試料としては、例えば、マイクロカプセルをろ過、遠心などの操作により分離した試料、試料中のマイクロカプセルを自然沈殿、凝集沈殿などで沈殿させた上清などを挙げることができるが、これに限定されることはない。あるいは、マイクロカプセル粒子表面の電荷による反応容器への吸着により、マイクロカプセルを試料から分離してもよい。マイクロカプセルの膜表面に電荷を持たせるには、膜表面にアミノ基もしくはカルボキシル基を付加するとよい。例えば、完成したアルギン酸ビーズ（マイクロカプセル）をアミノ基もしくはカルボキシル基を含む溶液に浸漬することにより電荷が付加されたビーズを得ることができる。アミノ基を含む溶液としては、メチルアミン、ピペリジン、スペルミン、スペルミジン、アニリン、ピリジン、トリエタノールアミン等の物質を含む溶液を例示することができる。カルボキシル基を含む溶液としては、不飽和カルボン酸（リノール酸、オレイン酸等）、ヒドロキシ酸（リンゴ酸、クエン酸等）、芳香族カルボン酸（フタル酸、安息香酸等）、ジカルボン酸（マロン酸、コハク酸）等の物質を含む溶液を例示することができる。あるいはまた、マイクロカプセルに磁性体を含有させることで、磁力による回収により、マイクロカプセルと試料を分離することができる。磁性体の例として、酸化鉄、コバルト、フェライト、酸化クロムなどが挙げられ、これらはビーズ（マイクロカプセル）の内側に封入するとよい。すなわち、酵素・色素（着色剤）・被膜となる物質と一緒に入れて滴下することで、磁性体をビーズの内側に封入することができる。
試料中に存在する細胞からATPを抽出するには、市販のATP抽出試薬を用いればよい。さらに、細胞から抽出されたATPは、ルシフェリン／ルシフェラーゼ反応により測定することができるが、この方法に限定されるわけではない。ルシフェリン／ルシフェラーゼ反応によるATPの検出は、市販のルシフェリン／ルシフェラーゼ発光試薬（LL発光試薬）を用いて発光させ、その発光をルミノメーターで測定することにより行うことができる。ルシフェリン／ルシフェラーゼ反応はATP量に依存する反応である。ルシフェリンとATPが反応し、アデニル酸ルシフェリンとなり、このアデニル酸ルシフェリンと酸素がルシフェラーゼ酵素の存在下で酸化的脱炭酸反応により分解され、この反応の過程において得られるエネルギーの一部が発光という反応として現れる。この発光を定量することでATPの定量を行うことができる。
ルシフェリン／ルシフェラーゼの発光反応系におけるルシフェラーゼの濃度は、0.1μg/mL～100μg/mLが適当であり、好ましくは1 μg/mL～20 μg/mLである。ルシフェラーゼは、甲虫由来のホタル・ルシフェリン、即ち多複素式有機酸Ｄ－（－）－２－（６‘ヒドロキシ－２’－ベンゾチアゾリル）－△２－チアゾリン－４－カルボン酸（以降は特に記載のない限り「ルシフェリン」と表記する）を発光基質とし、これを酸化触媒して光子を発する酵素で、ホタル科を始め、コメツキ科、ホタルモドキ科、イリオモテボタル科など甲虫由来で発光反応に与る酵素全てを含む。この中には組換えDNA技術や変異技術などにより、酵素タンパク自体の安定性や発光特性などが人為的に改変された酵素も含まれる。また、ルシフェリンの濃度は、0.001 mM～100 mMが適当であり、好ましくは0.01 mM～10 mMである。ルシフェリンは、甲虫由来のホタル・ルシフェリン（甲虫ルシフェリン）、即ち多複素式有機酸Ｄ－（－）－２－（６‘ヒドロキシ－２’－ベンゾチアゾリル）－△２－チアゾリン－４－カルボン酸）であるとよく、甲虫より直接抽出および精製されたものや、化学合成されたものを含む。さらには甲虫ルシフェリンの誘導体で、ある酵素の消化を受けた後に発光活性を持つ発光基質も含まれる。このような甲虫ルシフェリンの誘導体としては、4-メチル-D-ルシフェリン、D-ルシフェニル-L-メチオニン、６-O-ガラクトピラノシル-ルシフェリン、ＤＥＶＤ－ルシフェリン、ルシフェリン-6’メチルエステル、ルシフェリン6’-クロロエチルエステル、6’-デオキシルシフェリン、ルシフェリン6’ベンジルエステルなどを挙げることができる。ルシフェリンおよびその誘導体は塩の形態であってもよい。塩としては、カリウム塩、ナトリウム塩などを挙げることができる。 After the reaction, ATP is extracted from the cells present in the sample, and the extracted ATP is detected. The sample may be in a state that does not include microcapsules. Examples of the sample not containing microcapsules include a sample obtained by separating the microcapsule by an operation such as filtration and centrifugation, and a supernatant obtained by precipitating the microcapsule in the sample by natural precipitation or coagulation precipitation. However, the present invention is not limited to this. Or you may isolate | separate a microcapsule from a sample by adsorption | suction to the reaction container by the electric charge of the microcapsule particle surface. In order to give a charge to the membrane surface of the microcapsule, an amino group or a carboxyl group may be added to the membrane surface. For example, a charged bead can be obtained by immersing the completed alginate beads (microcapsules) in a solution containing an amino group or a carboxyl group. Examples of the solution containing an amino group include a solution containing a substance such as methylamine, piperidine, spermine, spermidine, aniline, pyridine, triethanolamine and the like. Examples of solutions containing carboxyl groups include unsaturated carboxylic acids (linoleic acid, oleic acid, etc.), hydroxy acids (malic acid, citric acid, etc.), aromatic carboxylic acids (phthalic acid, benzoic acid, etc.), dicarboxylic acids (malonic acid) And a solution containing a substance such as succinic acid). Alternatively, the microcapsule and the sample can be separated by collecting the magnetic material in the microcapsule and collecting it by magnetic force. Examples of the magnetic material include iron oxide, cobalt, ferrite, chromium oxide, and the like, and these may be enclosed inside a bead (microcapsule). That is, a magnetic substance can be enclosed inside a bead by dropping together with a substance that becomes an enzyme, a dye (colorant), and a film.
In order to extract ATP from cells present in the sample, a commercially available ATP extraction reagent may be used. Furthermore, ATP extracted from cells can be measured by a luciferin / luciferase reaction, but is not limited to this method. Detection of ATP by the luciferin / luciferase reaction can be performed by emitting light using a commercially available luciferin / luciferase luminescence reagent (LL luminescence reagent) and measuring the luminescence with a luminometer. The luciferin / luciferase reaction is a reaction that depends on the amount of ATP. Luciferin and ATP react to form adenylate luciferin, and this adenylate luciferin and oxygen are decomposed by oxidative decarboxylation in the presence of the luciferase enzyme, and a part of the energy obtained in the process of this reaction is luminescence. appear. By quantifying this luminescence, ATP can be quantified.
The concentration of luciferase in the luminescent reaction system luciferin / luciferase, 0.1 [mu] g / mL ~ 100 [mu] g / mL are suitable, preferably from 1 μg / mL ~ 20 μ g / mL. Luciferase is a firefly luciferin derived from a beetle, that is, a multi-heterocyclic organic acid D-(−)-2- (6′hydroxy-2′-benzothiazolyl) -Δ2-thiazoline-4-carboxylic acid (hereinafter specifically described) Unless otherwise indicated, it is expressed as “luciferin”). It is an enzyme that catalyzes oxidation and emits photons. It is an enzyme that emits photons. Contains all enzymes. This includes enzymes in which the stability and luminescence properties of the enzyme protein itself have been artificially altered by recombinant DNA technology or mutation technology. Further, the concentration of luciferin is suitably 0.001 mM to 100 mM, preferably 0.01 mM to 10 mM. Luciferin is a beetle-derived firefly luciferin (Coleoptera luciferin), that is, a multi-heterocyclic organic acid D-(−)-2- (6′hydroxy-2′-benzothiazolyl) -Δ2-thiazoline-4-carboxylic acid) Often this includes those extracted and purified directly from beetles and those that are chemically synthesized. Furthermore, a luminescent substrate which is a derivative of beetle luciferin and has luminescence activity after digestion with an enzyme. Such beetle luciferin derivatives include 4-methyl-D-luciferin, D-luciferyl-L-methionine, 6-O-galactopyranosyl-luciferin, DEVD-luciferin, luciferin-6'methyl ester, luciferin 6 Examples include '-chloroethyl ester, 6'-deoxyluciferin, and luciferin 6'benzyl ester. Luciferin and its derivatives may be in the form of a salt. Examples of the salt include potassium salt and sodium salt.
試料中に存在する細胞からATPを抽出するには、市販のATP抽出試薬を用いればよい。さらに、細胞から抽出されたATPは、ルシフェリン／ルシフェラーゼ反応により測定することができるが、この方法に限定されるわけではない。ルシフェリン／ルシフェラーゼ反応によるATPの検出は、市販のルシフェリン／ルシフェラーゼ発光試薬（LL発光試薬）を用いて発光させ、その発光をルミノメーターで測定することにより行うことができる。ルシフェリン／ルシフェラーゼ反応はATP量に依存する反応である。ルシフェリンとATPが反応し、アデニル酸ルシフェリンとなり、このアデニル酸ルシフェリンと酸素がルシフェラーゼ酵素の存在下で酸化的脱炭酸反応により分解され、この反応の過程において得られるエネルギーの一部が発光という反応として現れる。この発光を定量することでATPの定量を行うことができる。
ルシフェリン／ルシフェラーゼの発光反応系におけるルシフェラーゼの濃度は、0.1μg/mL～100μg/mLが適当であり、好ましくは1 μg/mL～20 μg/mLである。ルシフェラーゼは、甲虫由来のホタル・ルシフェリン、即ち多複素式有機酸Ｄ－（－）－２－（６‘ヒドロキシ－２’－ベンゾチアゾリル）－△２－チアゾリン－４－カルボン酸（以降は特に記載のない限り「ルシフェリン」と表記する）を発光基質とし、これを酸化触媒して光子を発する酵素で、ホタル科を始め、コメツキ科、ホタルモドキ科、イリオモテボタル科など甲虫由来で発光反応に与る酵素全てを含む。この中には組換えDNA技術や変異技術などにより、酵素タンパク自体の安定性や発光特性などが人為的に改変された酵素も含まれる。また、ルシフェリンの濃度は、0.001 mM～100 mMが適当であり、好ましくは0.01 mM～10 mMである。ルシフェリンは、甲虫由来のホタル・ルシフェリン（甲虫ルシフェリン）、即ち多複素式有機酸Ｄ－（－）－２－（６‘ヒドロキシ－２’－ベンゾチアゾリル）－△２－チアゾリン－４－カルボン酸）であるとよく、甲虫より直接抽出および精製されたものや、化学合成されたものを含む。さらには甲虫ルシフェリンの誘導体で、ある酵素の消化を受けた後に発光活性を持つ発光基質も含まれる。このような甲虫ルシフェリンの誘導体としては、4-メチル-D-ルシフェリン、D-ルシフェニル-L-メチオニン、６-O-ガラクトピラノシル-ルシフェリン、ＤＥＶＤ－ルシフェリン、ルシフェリン-6’メチルエステル、ルシフェリン6’-クロロエチルエステル、6’-デオキシルシフェリン、ルシフェリン6’ベンジルエステルなどを挙げることができる。ルシフェリンおよびその誘導体は塩の形態であってもよい。塩としては、カリウム塩、ナトリウム塩などを挙げることができる。 After the reaction, ATP is extracted from the cells present in the sample, and the extracted ATP is detected. The sample may be in a state that does not include microcapsules. Examples of the sample not containing microcapsules include a sample obtained by separating the microcapsule by an operation such as filtration and centrifugation, and a supernatant obtained by precipitating the microcapsule in the sample by natural precipitation or coagulation precipitation. However, the present invention is not limited to this. Or you may isolate | separate a microcapsule from a sample by adsorption | suction to the reaction container by the electric charge of the microcapsule particle surface. In order to give a charge to the membrane surface of the microcapsule, an amino group or a carboxyl group may be added to the membrane surface. For example, a charged bead can be obtained by immersing the completed alginate beads (microcapsules) in a solution containing an amino group or a carboxyl group. Examples of the solution containing an amino group include a solution containing a substance such as methylamine, piperidine, spermine, spermidine, aniline, pyridine, triethanolamine and the like. Examples of solutions containing carboxyl groups include unsaturated carboxylic acids (linoleic acid, oleic acid, etc.), hydroxy acids (malic acid, citric acid, etc.), aromatic carboxylic acids (phthalic acid, benzoic acid, etc.), dicarboxylic acids (malonic acid) And a solution containing a substance such as succinic acid). Alternatively, the microcapsule and the sample can be separated by collecting the magnetic material in the microcapsule and collecting it by magnetic force. Examples of the magnetic material include iron oxide, cobalt, ferrite, chromium oxide, and the like, and these may be enclosed inside a bead (microcapsule). That is, a magnetic substance can be enclosed inside a bead by dropping together with a substance that becomes an enzyme, a dye (colorant), and a film.
In order to extract ATP from cells present in the sample, a commercially available ATP extraction reagent may be used. Furthermore, ATP extracted from cells can be measured by a luciferin / luciferase reaction, but is not limited to this method. Detection of ATP by the luciferin / luciferase reaction can be performed by emitting light using a commercially available luciferin / luciferase luminescence reagent (LL luminescence reagent) and measuring the luminescence with a luminometer. The luciferin / luciferase reaction is a reaction that depends on the amount of ATP. Luciferin and ATP react to form adenylate luciferin, and this adenylate luciferin and oxygen are decomposed by oxidative decarboxylation in the presence of the luciferase enzyme, and a part of the energy obtained in the process of this reaction is luminescence. appear. By quantifying this luminescence, ATP can be quantified.
The concentration of luciferase in the luminescent reaction system luciferin / luciferase, 0.1 [mu] g / mL ~ 100 [mu] g / mL are suitable, preferably from 1 μg / mL ~ 20 μ g / mL. Luciferase is a firefly luciferin derived from a beetle, that is, a multi-heterocyclic organic acid D-(−)-2- (6′hydroxy-2′-benzothiazolyl) -Δ2-thiazoline-4-carboxylic acid (hereinafter specifically described) Unless otherwise indicated, it is expressed as “luciferin”). It is an enzyme that catalyzes oxidation and emits photons. It is an enzyme that emits photons. Contains all enzymes. This includes enzymes in which the stability and luminescence properties of the enzyme protein itself have been artificially altered by recombinant DNA technology or mutation technology. Further, the concentration of luciferin is suitably 0.001 mM to 100 mM, preferably 0.01 mM to 10 mM. Luciferin is a beetle-derived firefly luciferin (Coleoptera luciferin), that is, a multi-heterocyclic organic acid D-(−)-2- (6′hydroxy-2′-benzothiazolyl) -Δ2-thiazoline-4-carboxylic acid) Often this includes those extracted and purified directly from beetles and those that are chemically synthesized. Furthermore, a luminescent substrate which is a derivative of beetle luciferin and has luminescence activity after digestion with an enzyme. Such beetle luciferin derivatives include 4-methyl-D-luciferin, D-luciferyl-L-methionine, 6-O-galactopyranosyl-luciferin, DEVD-luciferin, luciferin-6'methyl ester, luciferin 6 Examples include '-chloroethyl ester, 6'-deoxyluciferin, and luciferin 6'benzyl ester. Luciferin and its derivatives may be in the form of a salt. Examples of the salt include potassium salt and sodium salt.
本発明の方法により、細胞由来のATPの定量、細胞の定量（生きている細胞の定量、既知の細胞であれば、高感度かつ正確に定量ができ、未知の細胞についても検出が可能）などができる。細胞は、微生物細胞、動物細胞、植物細胞などいかなる細胞であってもよい。本発明の方法は、食品衛生管理などで要求される微生物の検出・定量、洗浄度合の検査などに利用することができる。例えば、ルシフェリン／ルシフェラーゼ反応を利用することにより、10-16 molesのATPの検出が可能となり、細菌では約103個、酵母や真菌では数十個といった高感度の検出が可能となる。さらに、ATP除去から発光量測定/判定までに要する時間が1時間以内といった迅速な測定が可能となる。
With the method of the present invention, quantification of cell-derived ATP, quantification of cells (quantity of living cells, if known cells can be quantified with high sensitivity and accuracy, and unknown cells can also be detected), etc. Can do. The cell may be any cell such as a microbial cell, an animal cell, or a plant cell. The method of the present invention can be used for detection and quantification of microorganisms required for food hygiene management and the like, and inspection of the degree of cleaning. For example, by using the luciferin / luciferase reaction, 10 -16 moles of ATP can be detected, and high-sensitivity detection such as about 10 3 in bacteria and several tens in yeast and fungi is possible. Furthermore, it is possible to quickly measure that the time required from the ATP removal to the light emission amount measurement / judgment is within one hour.
また、本発明は、ATP分解酵素を内包するマイクロカプセルを提供する。
The present invention also provides a microcapsule that encapsulates ATP-degrading enzyme.
マイクロカプセルは、無機顔料として、二酸化チタン、亜鉛華（酸化亜鉛）、酸化鉄、酸化クロム、鉄黒、コバルトブルー、アルミナ白、酸化鉄黄、ビリジアン、硫化亜鉛、リトポン、カドミウムエロー、朱、カドミウムレッド、黄鉛、モリブデートオレンジ、ジンククロメート、ストロンチウムクロメート、 ホワイトカーボン、クレー、タルク、群青、沈降性硫酸バリウム、バライト粉、炭酸カルシウム、鉛白フェロシアン化物（紺青）、燐酸塩（マンガンバイオレット）、炭素（カーボンブラック）、有機顔料として、ローダミンレーキ、メチルバイオレットレーキ、キノリンエローレーキ、マラカイトグリーンレーキ、アリザリンレーキ、カーミン6B、レーキレットC、ジスアゾエロー、レーキレット4R、クロモフタルエロー3G、クロモフタルスカーレットRN 、ニッケルアゾエロー、パーマネントオレンジHL、フタロシアニンブルー、フタロシアニングリーン、フラバンスロンエロー、チオインジゴボルドー、ペリノンレッド、ジオキサドンバイオレット、キナクリドンレッド、ナフトールエローS、ピグロントグリーンB、ルモゲンエロー、シグナルレッド、アルカリブルー、アニリンブラックなどの着色剤、酵素保護剤として、牛血清アルブミン、ゼラチン、トレハロースなどを内包してもよい。
Microcapsules are inorganic pigments such as titanium dioxide, zinc white (zinc oxide), iron oxide, chromium oxide, iron black, cobalt blue, alumina white, iron oxide yellow, viridian, zinc sulfide, lithopone, cadmium yellow, vermilion, cadmium. Red, yellow lead, molybdate orange, zinc chromate, strontium chromate, agate white carbon, clay, talc, ultramarine, precipitated barium sulfate, barite powder, calcium carbonate, lead white ferrocyanide (bituminous), phosphate (manganese violet) , Carbon (carbon black), organic pigments, rhodamine lake, methyl violet lake, quinoline yellow lake, malachite green lake, alizarin lake, carmine 6B, lakeette C, disazo yellow, lakelet 4R, chromophthalo yellow 3G, black Phthascarlet RN, Nickel Azo Yellow, Permanent Orange HL, Phthalocyanine Blue, Phthalocyanine Green, Flavanthrone Yellow, Thioindigo Bordeaux, Perinone Red, Dioxadone Violet, Quinacridone Red, Naphthol Yellow S, Pigrant Green B, Lumogen Yellow, Signal Red, Bovine serum albumin, gelatin, trehalose or the like may be included as a colorant such as alkali blue or aniline black, or an enzyme protecting agent.
マイクロカプセルの材質、物性などについては上述した。
The material and physical properties of the microcapsule have been described above.
ATP分解酵素を内包するマイクロカプセルは、相分離法、液中乾燥法、融解分散冷却法、スプレードライ法、パンコーティング法、界面重合法、in situ重合法、液中硬化被覆法など公知のいかなる方法で製造してもよい。
Microcapsules containing ATP-degrading enzyme can be any known method such as phase separation, submerged drying, melt dispersion cooling, spray drying, pan coating, interfacial polymerization, in-situ polymerization, submerged curing coating. You may manufacture by the method.
ATP分解酵素を内包するマイクロカプセルを用いて、ATPを分解除去することができる。このマイクロカプセルにより、飲料中の原料由来のATPを除去し、その後に、試料中に存在する微生物などの細胞由来のATPを抽出し、測定することにより、試料中の細胞由来のATPを測定することができる。従って、本発明は、ATP分解酵素を内包するマイクロカプセルを含む、ATP測定試薬を提供する。
ATP can be decomposed and removed using microcapsules containing ATP-degrading enzyme. By using this microcapsule, ATP derived from the ingredients in the beverage is removed, and then ATP derived from cells such as microorganisms present in the sample is extracted and measured to measure ATP derived from cells in the sample. be able to. Therefore, the present invention provides an ATP measurement reagent comprising a microcapsule that encapsulates an ATP-degrading enzyme.
本発明のATP測定試薬において、ATP分解酵素内包カプセルは、凍結乾燥品、懸濁液（カプセル浮遊液）などのいかなる形態で包含されてもよい。懸濁液とする場合には、カプセルを適当な溶媒（例えば、トリス緩衝液、HEPES緩衝液、MES緩衝液、TES緩衝液、純水等）に分散させるとよい。
In the ATP measurement reagent of the present invention, the ATP-degrading enzyme-encapsulating capsule may be included in any form such as a lyophilized product or a suspension (capsule suspension). In the case of a suspension, the capsule may be dispersed in an appropriate solvent (for example, Tris buffer, HEPES buffer, MES buffer, TES buffer, pure water, etc.).
本発明のATP測定試薬には、さらに、pH調整剤、酸化防止剤、保存料、防かび剤などを含めてもよい。
The ATP measurement reagent of the present invention may further contain a pH adjuster, an antioxidant, a preservative, an antifungal agent and the like.
本発明のATP測定試薬は、ATPの測定、例えば、試料中に存在する細胞由来のATPの測定に利用することができる（上述）。従って、本発明は、上記のATP測定試薬を含む、ATP測定キットを提供する。
The ATP measurement reagent of the present invention can be used for ATP measurement, for example, measurement of cell-derived ATP present in a sample (described above). Therefore, the present invention provides an ATP measurement kit containing the above ATP measurement reagent.
本発明のATP測定キットには、さらに、ルシフェリン/ルシフェラーゼ発光試薬、発光試薬溶解液、ATP抽出剤、ATP標準試薬、発光量とATP量を対応づける検量線、取扱い説明書、ATP量を換算するソフトウエアなどを含めてもよい。
The ATP measurement kit of the present invention further converts a luciferin / luciferase luminescence reagent, a luminescence reagent solution, an ATP extractant, an ATP standard reagent, a calibration curve that associates the luminescence amount with the ATP amount, an instruction manual, and an ATP amount. Software etc. may be included.
以下、実施例に基づいて本発明を詳細に説明するが、本発明はこれらの実施例に限定されるものではない。
〔実施例１〕
Ｉ．ATP消去ビーズの調製方法
１．アピラーゼジャガイモ由来GradeIII（シグマ・アルドリッジ社）１．1 ml、アルギン酸ナトリウム（和光純薬）2 g、顔料インキ（グラフテック社 インクペン用水性顔料インク青）200 μlを純水200 mlに溶解した。
２．溶解液をマイクロビーズ作製装置（日本ビュッヒ社 Encapsulator B-390）にセットした。
３．マイクロビーズ作製装置に直径約150 μmの液滴を生成させた。
４．液滴を2%塩化カルシウム溶液中に滴下した。
５．表面がゲル化した、アピラーゼおよび顔料インキを含んだマイクロビーズを0.2 μm孔径のフィルター（ナルゲン フィルターユニット）を用いてろ過した。
６．フィルター上のろ物（マイクロカプセル）を10 mM HEPESバッファーで洗浄した。
７．マイクロカプセルを適当量の10 mM HEPESバッファーに再懸濁した。
８．上記操作で得られた上清100 μlに10-7mole/lのATP標準液（東洋ビーネット社）10μlを混合し、30分間反応させた。その後、LL発光試薬（菌士郎ATP発光試薬 LL100-1 東洋ビーネット社）100 μlを混合し30秒間反応させた後、その発光をルミノメーター（LB9507 ベルトールド社）で測定した。なお、測定の積算時間は10秒間とした。本操作により得られた結果と純水100 μlに10-7mole/lのATP標準液1 μlを添加したものの発光量を測定した結果を比較し、上清液の発光量が純水の発光量に対して95%以上であることを確認し、マイクロカプセル芯剤に含有されなかったアピラーゼが存在していないことを証明した。上清液とマイクロビーズ（ATP消去ビーズ）の外観を図２に示す。
マイクロビーズの物性値は、以下の通りである。
・平均粒度：155.2 um
・粒度分布：16.4%
（顕微鏡観察（倍率40）でカプセルの直径を実測して、平均粒度と粒度分布を決定した。１回の観察で100粒測定し、それを3回繰り返した。平均粒度は300粒の平均値、粒度分布はその標準偏差で示す。）
・比重：1.6（ピペット法（JIS Z8820-2:2004） に従い測定した。）
・１カプセル中のアピラーゼ含有量：11.6 ng（直径150umのカプセルの内側に含まれる液量は約1.8ulであるので、本実験の条件では11.6 ngの酵素が含まれていると計算される。尚、カプセルに内包させる酵素量は、カプセル形成前の溶液に投入する酵素量により調節が可能である。本実験では、200mlの溶液に1.1mgのアピラーゼを混合したが、例えば、110mgのアピラーゼを混合した場合は、内包酵素量は110ngと推定される。> EXAMPLES Hereinafter, although this invention is demonstrated in detail based on an Example, this invention is not limited to these Examples.
[Example 1]
I. Method for preparing ATP-erased beads 1.1 ml of apyrase potato-derived Grade III (Sigma Aldridge), 2 g of sodium alginate (Wako Pure Chemical Industries), and 200 μl of pigment ink (aqueous pigment ink blue for graph pen ink pen) were dissolved in 200 ml of pure water.
2. The lysate was set in a microbead manufacturing apparatus (Nippon Büch Encapsulator B-390).
3. A droplet having a diameter of about 150 μm was generated on the microbead manufacturing apparatus.
4). The droplet was dropped into a 2% calcium chloride solution.
5. The microbeads containing apyrase and pigment ink whose surface was gelled were filtered using a 0.2 μm pore size filter (Nalgen filter unit).
6). The filtrate (microcapsule) on the filter was washed with 10 mM HEPES buffer.
7). The microcapsules were resuspended in an appropriate amount of 10 mM HEPES buffer.
8). 10 μl of a 10 −7 mole / l ATP standard solution (Toyo Benet) was mixed with 100 μl of the supernatant obtained by the above operation, and reacted for 30 minutes. Then, after mixing 100 microliters of LL luminescent reagent (Fujiro ATP luminescent reagent LL100-1 Toyo Beenet) and making it react for 30 seconds, the luminescence was measured with the luminometer (LB9507 Berthold). The integration time for measurement was 10 seconds. The result obtained by this operation is compared with the result of measuring the amount of luminescence of 1 μl of 10 -7 mole / l ATP standard solution added to 100 μl of pure water. It was confirmed that the amount was 95% or more, and it was proved that there was no apyrase that was not contained in the microcapsule core. The appearance of the supernatant and microbeads (ATP-erased beads) are shown in FIG.
The physical property values of the microbeads are as follows.
・ Average particle size: 155.2 um
・ Particle size distribution: 16.4%
(The microscopic observation (magnification 40) measured the capsule diameter to determine the average particle size and particle size distribution. 100 particles were measured in one observation, and this was repeated three times. The average particle size was the average value of 300 particles. The particle size distribution is indicated by its standard deviation.)
Specific gravity: 1.6 (Measured according to the pipette method (JIS Z8820-2: 2004).)
-Apyrase content in one capsule: 11.6 ng (The amount of liquid contained inside a capsule having a diameter of 150 um is about 1.8 ul, so it is calculated that 11.6 ng of enzyme is contained in the conditions of this experiment. The amount of enzyme contained in the capsule can be adjusted by the amount of enzyme added to the solution before capsule formation.In this experiment, 1.1 mg of apyrase was mixed with 200 ml of solution. When mixed, the amount of encapsulated enzyme is estimated to be 110 ng.>
〔実施例１〕
Ｉ．ATP消去ビーズの調製方法
１．アピラーゼジャガイモ由来GradeIII（シグマ・アルドリッジ社）１．1 ml、アルギン酸ナトリウム（和光純薬）2 g、顔料インキ（グラフテック社 インクペン用水性顔料インク青）200 μlを純水200 mlに溶解した。
２．溶解液をマイクロビーズ作製装置（日本ビュッヒ社 Encapsulator B-390）にセットした。
３．マイクロビーズ作製装置に直径約150 μmの液滴を生成させた。
４．液滴を2%塩化カルシウム溶液中に滴下した。
５．表面がゲル化した、アピラーゼおよび顔料インキを含んだマイクロビーズを0.2 μm孔径のフィルター（ナルゲン フィルターユニット）を用いてろ過した。
６．フィルター上のろ物（マイクロカプセル）を10 mM HEPESバッファーで洗浄した。
７．マイクロカプセルを適当量の10 mM HEPESバッファーに再懸濁した。
８．上記操作で得られた上清100 μlに10-7mole/lのATP標準液（東洋ビーネット社）10μlを混合し、30分間反応させた。その後、LL発光試薬（菌士郎ATP発光試薬 LL100-1 東洋ビーネット社）100 μlを混合し30秒間反応させた後、その発光をルミノメーター（LB9507 ベルトールド社）で測定した。なお、測定の積算時間は10秒間とした。本操作により得られた結果と純水100 μlに10-7mole/lのATP標準液1 μlを添加したものの発光量を測定した結果を比較し、上清液の発光量が純水の発光量に対して95%以上であることを確認し、マイクロカプセル芯剤に含有されなかったアピラーゼが存在していないことを証明した。上清液とマイクロビーズ（ATP消去ビーズ）の外観を図２に示す。
マイクロビーズの物性値は、以下の通りである。
・平均粒度：155.2 um
・粒度分布：16.4%
（顕微鏡観察（倍率40）でカプセルの直径を実測して、平均粒度と粒度分布を決定した。１回の観察で100粒測定し、それを3回繰り返した。平均粒度は300粒の平均値、粒度分布はその標準偏差で示す。）
・比重：1.6（ピペット法（JIS Z8820-2:2004） に従い測定した。）
・１カプセル中のアピラーゼ含有量：11.6 ng（直径150umのカプセルの内側に含まれる液量は約1.8ulであるので、本実験の条件では11.6 ngの酵素が含まれていると計算される。尚、カプセルに内包させる酵素量は、カプセル形成前の溶液に投入する酵素量により調節が可能である。本実験では、200mlの溶液に1.1mgのアピラーゼを混合したが、例えば、110mgのアピラーゼを混合した場合は、内包酵素量は110ngと推定される。> EXAMPLES Hereinafter, although this invention is demonstrated in detail based on an Example, this invention is not limited to these Examples.
[Example 1]
I. Method for preparing ATP-erased beads 1.1 ml of apyrase potato-derived Grade III (Sigma Aldridge), 2 g of sodium alginate (Wako Pure Chemical Industries), and 200 μl of pigment ink (aqueous pigment ink blue for graph pen ink pen) were dissolved in 200 ml of pure water.
2. The lysate was set in a microbead manufacturing apparatus (Nippon Büch Encapsulator B-390).
3. A droplet having a diameter of about 150 μm was generated on the microbead manufacturing apparatus.
4). The droplet was dropped into a 2% calcium chloride solution.
5. The microbeads containing apyrase and pigment ink whose surface was gelled were filtered using a 0.2 μm pore size filter (Nalgen filter unit).
6). The filtrate (microcapsule) on the filter was washed with 10 mM HEPES buffer.
7). The microcapsules were resuspended in an appropriate amount of 10 mM HEPES buffer.
8). 10 μl of a 10 −7 mole / l ATP standard solution (Toyo Benet) was mixed with 100 μl of the supernatant obtained by the above operation, and reacted for 30 minutes. Then, after mixing 100 microliters of LL luminescent reagent (Fujiro ATP luminescent reagent LL100-1 Toyo Beenet) and making it react for 30 seconds, the luminescence was measured with the luminometer (LB9507 Berthold). The integration time for measurement was 10 seconds. The result obtained by this operation is compared with the result of measuring the amount of luminescence of 1 μl of 10 -7 mole / l ATP standard solution added to 100 μl of pure water. It was confirmed that the amount was 95% or more, and it was proved that there was no apyrase that was not contained in the microcapsule core. The appearance of the supernatant and microbeads (ATP-erased beads) are shown in FIG.
The physical property values of the microbeads are as follows.
・ Average particle size: 155.2 um
・ Particle size distribution: 16.4%
(The microscopic observation (magnification 40) measured the capsule diameter to determine the average particle size and particle size distribution. 100 particles were measured in one observation, and this was repeated three times. The average particle size was the average value of 300 particles. The particle size distribution is indicated by its standard deviation.)
Specific gravity: 1.6 (Measured according to the pipette method (JIS Z8820-2: 2004).)
-Apyrase content in one capsule: 11.6 ng (The amount of liquid contained inside a capsule having a diameter of 150 um is about 1.8 ul, so it is calculated that 11.6 ng of enzyme is contained in the conditions of this experiment. The amount of enzyme contained in the capsule can be adjusted by the amount of enzyme added to the solution before capsule formation.In this experiment, 1.1 mg of apyrase was mixed with 200 ml of solution. When mixed, the amount of encapsulated enzyme is estimated to be 110 ng.>
II．ATP消去ビーズ 洗浄操作の必要性
Ｉ．（ATP消去ビーズの調製方法）の７．のマイクロカプセル懸濁液（表１では、「マイクロカプセル含有液」と表示）とIの８．の上清液の各々100 μlに10-7mole/lのATP標準液（東洋ビーネット社）10μlを混合し、30分間反応させた。その後、LL発光試薬（菌士郎ATP発光試薬 LL100-1 東洋ビーネット社）100 μlを混合し30秒間反応させた後、その発光をルミノメーター（LB9507 ベルトールド社）で測定した。なお、測定の積算時間は10秒間とした。結果を表１にまとめた（表１の「洗浄操作あり」）
また、Ｉ．（ATP消去ビーズの調製方法）の５．６．７を省略した場合についても、同様の測定を行い、結果を表１にまとめた（表１の「未洗浄」）。「未洗浄」の場合、上清液にマイクロカプセルに包摂されなかったと考えられるアピラーゼが残存し、これがATPを分解した結果、本来得られるはずである発光量を2桁下げていると考えられる。このことは、本来の目的物である抽出した微生物由来のATPが上清液に残存しているアピラーゼにより分解されてしまう可能性を示唆する。このため、マイクロカプセルをろ過にて回収後HEPESバッファーで洗浄する操作により、未包摂アピラーゼを除去することが好ましいと考えられる。なお、表1におけるマイクロカプセル含有液はカプセル中にアピラーゼを含んでいるのでATP分解活性がある。 II. ATP-erased beads Necessity of washing operation 6. (Method for preparing ATP-erased beads) Microcapsule suspension (indicated as “microcapsule-containing liquid” in Table 1) and I.8. 10 μl of a 10 −7 mole / l ATP standard solution (Toyo Benet Corporation) was mixed with 100 μl of each of the supernatants, and reacted for 30 minutes. Then, after mixing 100 microliters of LL luminescent reagent (Fujiro ATP luminescent reagent LL100-1 Toyo Beenet) and making it react for 30 seconds, the luminescence was measured with the luminometer (LB9507 Berthold). The integration time for measurement was 10 seconds. The results are summarized in Table 1 ("With cleaning operation" in Table 1)
In addition, I.I. In the case where 5.6.7 in (Method for preparing ATP-erased beads) was omitted, the same measurement was performed, and the results are summarized in Table 1 (“Unwashed” in Table 1). In the case of “unwashed”, it is considered that apyrase, which was considered not to be encapsulated in the microcapsule, remained in the supernatant, and this reduced ATP by 2 orders of magnitude as a result of decomposing ATP. This suggests that ATP derived from the extracted microorganism, which is the original target product, may be degraded by the apyrase remaining in the supernatant. For this reason, it is considered preferable to remove unencapsulated apyrase by an operation of recovering the microcapsules by filtration and washing with a HEPES buffer. In addition, since the microcapsule-containing liquid in Table 1 contains apyrase in the capsule, it has ATP degradation activity.
Ｉ．（ATP消去ビーズの調製方法）の７．のマイクロカプセル懸濁液（表１では、「マイクロカプセル含有液」と表示）とIの８．の上清液の各々100 μlに10-7mole/lのATP標準液（東洋ビーネット社）10μlを混合し、30分間反応させた。その後、LL発光試薬（菌士郎ATP発光試薬 LL100-1 東洋ビーネット社）100 μlを混合し30秒間反応させた後、その発光をルミノメーター（LB9507 ベルトールド社）で測定した。なお、測定の積算時間は10秒間とした。結果を表１にまとめた（表１の「洗浄操作あり」）
また、Ｉ．（ATP消去ビーズの調製方法）の５．６．７を省略した場合についても、同様の測定を行い、結果を表１にまとめた（表１の「未洗浄」）。「未洗浄」の場合、上清液にマイクロカプセルに包摂されなかったと考えられるアピラーゼが残存し、これがATPを分解した結果、本来得られるはずである発光量を2桁下げていると考えられる。このことは、本来の目的物である抽出した微生物由来のATPが上清液に残存しているアピラーゼにより分解されてしまう可能性を示唆する。このため、マイクロカプセルをろ過にて回収後HEPESバッファーで洗浄する操作により、未包摂アピラーゼを除去することが好ましいと考えられる。なお、表1におけるマイクロカプセル含有液はカプセル中にアピラーゼを含んでいるのでATP分解活性がある。 II. ATP-erased beads Necessity of washing operation 6. (Method for preparing ATP-erased beads) Microcapsule suspension (indicated as “microcapsule-containing liquid” in Table 1) and I.8. 10 μl of a 10 −7 mole / l ATP standard solution (Toyo Benet Corporation) was mixed with 100 μl of each of the supernatants, and reacted for 30 minutes. Then, after mixing 100 microliters of LL luminescent reagent (Fujiro ATP luminescent reagent LL100-1 Toyo Beenet) and making it react for 30 seconds, the luminescence was measured with the luminometer (LB9507 Berthold). The integration time for measurement was 10 seconds. The results are summarized in Table 1 ("With cleaning operation" in Table 1)
In addition, I.I. In the case where 5.6.7 in (Method for preparing ATP-erased beads) was omitted, the same measurement was performed, and the results are summarized in Table 1 (“Unwashed” in Table 1). In the case of “unwashed”, it is considered that apyrase, which was considered not to be encapsulated in the microcapsule, remained in the supernatant, and this reduced ATP by 2 orders of magnitude as a result of decomposing ATP. This suggests that ATP derived from the extracted microorganism, which is the original target product, may be degraded by the apyrase remaining in the supernatant. For this reason, it is considered preferable to remove unencapsulated apyrase by an operation of recovering the microcapsules by filtration and washing with a HEPES buffer. In addition, since the microcapsule-containing liquid in Table 1 contains apyrase in the capsule, it has ATP degradation activity.
対照区は、HEPESバッファー100 μlに10-7mole/lのATP標準液（東洋ビーネット社）10μlを混合し、30分間反応させた。その後、LL発光試薬（菌士郎ATP発光試薬 LL100-1 東洋ビーネット社）100 μlを混合し30秒間反応させた後、その発光をルミノメーター（LB9507 ベルトールド社）で測定した（測定の積算時間は10秒間）。
In the control group, 10 μl of 10 −7 mole / l ATP standard solution (Toyo B-Net) was mixed with 100 μl of HEPES buffer and reacted for 30 minutes. After that, after mixing 100 μl of LL luminescence reagent (Fujiro ATP luminescence reagent LL100-1 Toyo Benet Corporation) and reacting for 30 seconds, the luminescence was measured with a luminometer (LB9507 Berthold). 10 seconds).
III．サンプル中のATP消去・サンプル中の微生物由来ATP量の測定方法（一例）
１．検体（緑茶・ジュース・ATP標準サンプルなど）200 μlとマイクロビーズ含有液200 μlを混合し、室温で30分間放置した。この間にマイクロビーズが沈殿した。
２．反応後、上清100 μlを分取し、等量のATP抽出試薬（菌士郎ATP抽出試薬 LL100-2 東洋ビーネット社）を添加した。30秒間反応させた。
３．上記２の反応液に200 μlのLL発光試薬（菌士郎ATP発光試薬 LL100-1 東洋ビーネット社）を添加し、その発光をルミノメーターで測定した。 III. Eliminating ATP in a sample / Measuring method of microorganism-derived ATP in a sample (example)
1. 200 μl of a sample (green tea, juice, ATP standard sample, etc.) and 200 μl of a microbead-containing solution were mixed and left at room temperature for 30 minutes. During this time, microbeads precipitated.
2. After the reaction, 100 μl of the supernatant was collected, and an equal amount of ATP extraction reagent (Fujiro Shiro ATP extraction reagent LL100-2 Toyo B-Net) was added. Reacted for 30 seconds.
3. 200 μl of LL luminescence reagent (Fujiro ATP luminescence reagent LL100-1 Toyo B-Net) was added to the above reaction solution 2, and the luminescence was measured with a luminometer.
１．検体（緑茶・ジュース・ATP標準サンプルなど）200 μlとマイクロビーズ含有液200 μlを混合し、室温で30分間放置した。この間にマイクロビーズが沈殿した。
２．反応後、上清100 μlを分取し、等量のATP抽出試薬（菌士郎ATP抽出試薬 LL100-2 東洋ビーネット社）を添加した。30秒間反応させた。
３．上記２の反応液に200 μlのLL発光試薬（菌士郎ATP発光試薬 LL100-1 東洋ビーネット社）を添加し、その発光をルミノメーターで測定した。 III. Eliminating ATP in a sample / Measuring method of microorganism-derived ATP in a sample (example)
1. 200 μl of a sample (green tea, juice, ATP standard sample, etc.) and 200 μl of a microbead-containing solution were mixed and left at room temperature for 30 minutes. During this time, microbeads precipitated.
2. After the reaction, 100 μl of the supernatant was collected, and an equal amount of ATP extraction reagent (Fujiro Shiro ATP extraction reagent LL100-2 Toyo B-Net) was added. Reacted for 30 seconds.
3. 200 μl of LL luminescence reagent (Fujiro ATP luminescence reagent LL100-1 Toyo B-Net) was added to the above reaction solution 2, and the luminescence was measured with a luminometer.
IV．ビーズ処理による緑茶中のATP分解の経時変化
IIIの測定結果（検体：緑茶）を下記の表２に示す。 IV. Table 2 below shows the measurement results of III of ATP degradation in green tea by bead treatment III (specimen: green tea).
IIIの測定結果（検体：緑茶）を下記の表２に示す。 IV. Table 2 below shows the measurement results of III of ATP degradation in green tea by bead treatment III (specimen: green tea).
緑茶に含まれているATPをマイクロカプセル法および従来法（菌士郎ATP測定キット（東洋ビーネット）の手順に従う方法）により分解を試みた。マイクロカプセル法は、緑茶200 μlに対して200 μlのマイクロカプセル液を添加し、表2における処理時間、室温下に静置した。時間経過後、100 μlのサンプル液を分取して等量100 μlのATP発光試薬（東洋ビーネット）を添加してその発光量を測定した。従来法においては、緑茶200 μlに対してATP除去試薬（東洋ビーネット）200 μlを添加して表2の処理時間室温下に静置した。経過後、100 μlのサンプルを分取、100 μlのATP発光試薬（東洋ビーネット）を添加して発光量を測定した。マイクロカプセル法では、30分の処理でバックグラウンドレベルまで発光量を低減できているので緑茶由来ATPの分解が完了しているのに対し、従来法では60分の処理でも発光が確認されるため、ATPの除去が不十分であると考えられる。
ATP contained in green tea was attempted to be decomposed by the microcapsule method and the conventional method (method according to the procedure of the Mycosis ATP measurement kit (Toyo B-Net)). In the microcapsule method, 200 μl of microcapsule solution was added to 200 μl of green tea, and the mixture was allowed to stand at room temperature for the treatment time shown in Table 2. After the elapse of time, 100 μl of the sample solution was fractionated, and an equal amount of 100 μl of ATP luminescence reagent (Toyo Benet) was added to measure the amount of luminescence. In the conventional method, 200 μl of an ATP removal reagent (Toyo Benet) was added to 200 μl of green tea and allowed to stand at room temperature for the treatment times shown in Table 2. After the lapse of time, 100 μl of a sample was collected, 100 μl of ATP luminescence reagent (Toyo Benet) was added, and the amount of luminescence was measured. In the microcapsule method, the amount of luminescence can be reduced to the background level in 30 minutes, so the decomposition of green tea-derived ATP has been completed, whereas in the conventional method, luminescence is confirmed even in 60 minutes. , ATP removal is considered insufficient.
V．検体（緑茶）中の大腸菌検出例
検体（緑茶）1 mlに大腸菌 5 x 105個を混入してその存在をATP量の変化で検出した結果を下記の表３に示す。 V. Example of detection of Escherichia coli in a sample (green tea) Table 3 below shows the results of detecting the presence of 5 × 10 5 E. coli cells in 1 ml of the sample (green tea) by detecting the change in the amount of ATP.
検体（緑茶）1 mlに大腸菌 5 x 105個を混入してその存在をATP量の変化で検出した結果を下記の表３に示す。 V. Example of detection of Escherichia coli in a sample (green tea) Table 3 below shows the results of detecting the presence of 5 × 10 5 E. coli cells in 1 ml of the sample (green tea) by detecting the change in the amount of ATP.
マイクロカプセル法の微生物混入試験では、大腸菌を混入緑茶飲料200 μlをチューブに分取し、200 μlのアピラーゼ含有マイクロカプセル懸濁液200 μlと混合して室温下30分間静置した。上澄み液100 μlを、マイクロカプセルが混入しないよう注意して回収した。次に、回収した溶液に対して等量である100 μlのATP抽出試薬（菌士郎ATP抽出試薬LL100-2 東洋ビーネット）を添加して10秒間微生物からのATP抽出処理を行った。抽出されたATPの定量を行うため、200 mlのATP発光試薬（菌士郎 ATP発光試薬 LL100-1）を加え、ルミノメーター（LB9507 ベルトールド社）にチューブをセットして10秒間の積算発光量を得た。
In the microcapsule microbe contamination test, 200 μl of green tea beverage mixed with E. coli was dispensed into a tube, mixed with 200 μl of 200 μl of apyrase-containing microcapsule suspension, and allowed to stand at room temperature for 30 minutes. 100 μl of the supernatant was collected taking care not to contaminate the microcapsules. Next, an equal amount of 100 μl of ATP extraction reagent (Fujiro Shiro ATP extraction reagent LL100-2, Toyo Benet) was added to the collected solution, and ATP extraction from microorganisms was performed for 10 seconds. To quantitate the extracted ATP, add 200 ml ATP luminescence reagent (Fujiro Shiro ATP luminescence reagent LL100-1) and set the tube in a luminometer (LB9507 Berthold) to obtain the accumulated luminescence for 10 seconds. It was.
従来法は菌士郎ATP測定キットの手順に従った。すなわち、大腸菌を混入緑茶飲料200 μlをチューブに分取し、200 μlのATP除去試薬（菌士郎ATP除去試薬 LL100-3）と混合して室温下30分間静置した。上澄み液100 μlを回収し、その溶液に対して等量である100 μlのATP抽出試薬（菌士郎ATP抽出試薬LL100-2 東洋ビーネット）を添加して10秒間微生物からのATP抽出処理を行った。抽出されたATPの定量を行うため、200 mlのATP発光試薬（菌士郎 ATP発光試薬 LL100-1）を加え、ルミノメーター（LB9507 ベルトールド社）にチューブをセットして10秒間の積算発光量を得た。
The conventional method followed the procedure of the Mycosis ATP measurement kit. That is, 200 μl of green tea beverage mixed with Escherichia coli was collected in a tube, mixed with 200 μl of ATP removal reagent (Fujiro ATP removal reagent LL100-3), and allowed to stand at room temperature for 30 minutes. Collect 100 µl of the supernatant and add 100 µl of an equal amount of ATP extraction reagent (Fujiro ATP extraction reagent LL100-2, Toyo Benet) to the solution, and perform ATP extraction from microorganisms for 10 seconds. It was. To quantitate the extracted ATP, add 200 ml ATP luminescence reagent (Fujiro Shiro ATP luminescence reagent LL100-1) and set the tube in a luminometer (LB9507 Berthold) to obtain the accumulated luminescence for 10 seconds. It was.
従来法では大腸菌混入サンプルと無菌サンプル（菌混入なし）との発光量の差が、桁が変わらずわずかで、微生物由来のATPの有無の判断が困難となっている。対して、マクロカプセル法では緑茶由来のATPを高効率で消去できるため、大腸菌から抽出されたと考えられるATP由来の生物発光がバックグラウンドレベル（無菌サンプルの発光量）と比べ200倍であった。
本明細書で引用した全ての刊行物、特許および特許出願をそのまま参考として本明細書にとり入れるものとする。 In the conventional method, the difference in the amount of luminescence between the E. coli-contaminated sample and the aseptic sample (no microbial contamination) remains the same without changing the digit, making it difficult to determine the presence or absence of microorganism-derived ATP. On the other hand, since ATP derived from green tea can be erased with high efficiency by the macrocapsule method, the bioluminescence derived from ATP, which is thought to be extracted from Escherichia coli, was 200 times higher than the background level (the amount of luminescence of sterile samples).
All publications, patents and patent applications cited herein are incorporated herein by reference in their entirety.
本明細書で引用した全ての刊行物、特許および特許出願をそのまま参考として本明細書にとり入れるものとする。 In the conventional method, the difference in the amount of luminescence between the E. coli-contaminated sample and the aseptic sample (no microbial contamination) remains the same without changing the digit, making it difficult to determine the presence or absence of microorganism-derived ATP. On the other hand, since ATP derived from green tea can be erased with high efficiency by the macrocapsule method, the bioluminescence derived from ATP, which is thought to be extracted from Escherichia coli, was 200 times higher than the background level (the amount of luminescence of sterile samples).
All publications, patents and patent applications cited herein are incorporated herein by reference in their entirety.
本発明は、飲料などの食品中の微生物汚染の測定に用いることができる。
The present invention can be used for measuring microbial contamination in foods such as beverages.
Claims (8)
- アデノシン三リン酸分解酵素を内包するマイクロカプセルを用いて、試料中の遊離アデノシン三リン酸を分解した後、マイクロカプセルを含まない試料中に存在する細胞からアデノシン三リン酸を抽出し、抽出されたアデノシン三リン酸を測定することを含む、細胞由来のアデノシン三リン酸を測定する方法。 After degrading free adenosine triphosphate in the sample using a microcapsule encapsulating adenosine triphosphate degrading enzyme, adenosine triphosphate is extracted from the cells present in the sample that does not contain the microcapsule. A method for measuring cell-derived adenosine triphosphate, comprising measuring adenosine triphosphate.
- アデノシン三リン酸分解酵素が、アピラーゼ、酸性ホスファターゼ、アルカリホスファターゼ、ヘキソキナーゼ、ヌクレアーゼＰ及びデアミナーゼからなる群より選択される少なくとも１種の酵素である請求項１記載の方法。 The method according to claim 1, wherein the adenosine triphosphate degrading enzyme is at least one enzyme selected from the group consisting of apyrase, acid phosphatase, alkaline phosphatase, hexokinase, nuclease P and deaminase.
- マイクロカプセルの被膜が、分子量1000以下のイオンは透過させ、アデノシン三リン酸分解酵素は透過しない半透膜である請求項１又は２記載の方法。 The method according to claim 1 or 2, wherein the microcapsule film is a semipermeable membrane that allows ions having a molecular weight of 1000 or less to pass therethrough and does not allow adenosine triphosphate degrading enzyme to pass through.
- マイクロカプセルの被膜が、アルギン酸、メトキシペクチン、硫酸セルロース、ゼラチン、キトサン、カラギーナン及びワックスからなる群より選択される少なくとも１種の材料を含む請求項１～３のいずれかに記載の方法。 The method according to any one of claims 1 to 3, wherein the film of the microcapsule comprises at least one material selected from the group consisting of alginic acid, methoxy pectin, cellulose sulfate, gelatin, chitosan, carrageenan and wax.
- １カプセル中のアデノシン三リン酸分解酵素含有量が0.1 ng～1 mgである請求項１～４のいずれかに記載の方法。 The method according to any one of claims 1 to 4, wherein the content of adenosine triphosphate degrading enzyme in one capsule is 0.1 to 1 mg.
- ルシフェリン／ルシフェラーゼ反応により、アデノシン三リン酸を測定する請求項１～５のいずれかに記載の方法。 The method according to any one of claims 1 to 5, wherein adenosine triphosphate is measured by a luciferin / luciferase reaction.
- アデノシン三リン酸分解酵素を内包するマイクロカプセルを含む、アデノシン三リン酸測定試薬。 A reagent for measuring adenosine triphosphate comprising a microcapsule encapsulating adenosine triphosphate degrading enzyme.
- 請求項７記載のアデノシン三リン酸測定試薬を含む、アデノシン三リン酸測定キット。 An adenosine triphosphate measurement kit comprising the adenosine triphosphate measurement reagent according to claim 7.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| JPS6156076A (en) * | 1984-08-25 | 1986-03-20 | Agency Of Ind Science & Technol | Immobilized enzyme and its preparation |
| JP2003210197A (en) * | 2002-01-22 | 2003-07-29 | Unitika Ltd | Method for removing atp in sample solution |
| JP2009178091A (en) * | 2008-01-31 | 2009-08-13 | National Institute Of Advanced Industrial & Technology | Enzyme-including inorganic microcapsule, method for producing the same and use thereof |
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| JPH1156393A (en) * | 1997-08-21 | 1999-03-02 | Kikkoman Corp | Atp assay and reagent kit therefor |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS6156076A (en) * | 1984-08-25 | 1986-03-20 | Agency Of Ind Science & Technol | Immobilized enzyme and its preparation |
| JP2003210197A (en) * | 2002-01-22 | 2003-07-29 | Unitika Ltd | Method for removing atp in sample solution |
| JP2009178091A (en) * | 2008-01-31 | 2009-08-13 | National Institute Of Advanced Industrial & Technology | Enzyme-including inorganic microcapsule, method for producing the same and use thereof |
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