WO2002092202A1 - Procede pour traiter une membrane filtrante, membrane filtrante et procede pour compter rapidement un nombre de micro-organismes a l'aide de cette membrane filtrante - Google Patents

Procede pour traiter une membrane filtrante, membrane filtrante et procede pour compter rapidement un nombre de micro-organismes a l'aide de cette membrane filtrante Download PDF

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Publication number
WO2002092202A1
WO2002092202A1 PCT/JP2002/004733 JP0204733W WO02092202A1 WO 2002092202 A1 WO2002092202 A1 WO 2002092202A1 JP 0204733 W JP0204733 W JP 0204733W WO 02092202 A1 WO02092202 A1 WO 02092202A1
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WIPO (PCT)
Prior art keywords
hypoiodite
hypobromite
hypochlorite
filtration membrane
membrane
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PCT/JP2002/004733
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English (en)
Japanese (ja)
Inventor
Tatsuya Sakakibara
Toshinori Igarashi
Seiji Murakami
Yoshio Monji
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Kikkoman Corporation
Nihon Millipore K.K.
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Publication of WO2002092202A1 publication Critical patent/WO2002092202A1/fr

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D67/00—Processes specially adapted for manufacturing semi-permeable membranes for separation processes or apparatus
    • B01D67/0081—After-treatment of organic or inorganic membranes
    • B01D67/0088—Physical treatment with compounds, e.g. swelling, coating or impregnation
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D65/00—Accessories or auxiliary operations, in general, for separation processes or apparatus using semi-permeable membranes
    • B01D65/02—Membrane cleaning or sterilisation ; Membrane regeneration
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • C12Q1/06—Quantitative determination
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D2321/00—Details relating to membrane cleaning, regeneration, sterilization or to the prevention of fouling
    • B01D2321/16—Use of chemical agents
    • B01D2321/162—Use of acids
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D2321/00—Details relating to membrane cleaning, regeneration, sterilization or to the prevention of fouling
    • B01D2321/16—Use of chemical agents
    • B01D2321/164—Use of bases
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D2321/00—Details relating to membrane cleaning, regeneration, sterilization or to the prevention of fouling
    • B01D2321/16—Use of chemical agents
    • B01D2321/166—Use of enzymatic agents
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D2321/00—Details relating to membrane cleaning, regeneration, sterilization or to the prevention of fouling
    • B01D2321/16—Use of chemical agents
    • B01D2321/168—Use of other chemical agents

Definitions

  • the present invention relates to a method for treating a filtration membrane or a water absorption zone. Further, the present invention relates to a method for measuring microorganisms in a sample solution quickly, simply, and with high sensitivity. More specifically, the present invention relates to a method for measuring microorganisms captured on a filtration membrane by using an ATP conversion reaction reagent and a bioluminescent reagent. Background art
  • a rapid detection method of bacteria For example, if the number of viable bacteria in the sample solution is sufficiently large, a small amount should be collected in a small test tube. If the number of viable bacteria is slightly small, the sample solution should be filtered through a filtration membrane to capture microorganisms. After the filter membrane is immersed in a very small amount of sterile water or the like and a part of the liquid in which the microorganisms are suspended is collected in a small test tube.
  • the present applicants first filtered a sample solution containing microorganisms through a filtration membrane, captured the microorganisms in the sample solution on the filtration membrane, extracted biological components in the microorganisms,
  • a membrane filter comprising a number of small hydrophilic filtration membrane sections surrounded by a number of hydrophobic compartment walls described in Patent No. 3133328, and JP-A-6-237793.
  • the hydrophobic filter membrane described in the item (1) adheres to the point of attachment of live bacteria in ultrafine particles due to surface tension. It is excellent in that the extracted luminescent component is not diffused but is held in the form of ultrafine particles to emit light.
  • An object of the present invention is to provide a rapid, simple, and highly sensitive method for measuring microorganisms captured on a filtration membrane.
  • an object of the present invention is to prevent contamination of adenosine phosphates such as AMP, ATP or RNA, which is a cause of noise luminescent spots and background luminescence, appearing on these membranes in the above-described measurement method using a bioluminescence reaction. By eliminating them, the appearance of noise luminescent spots and background luminescence is eliminated, and more accurate and rapid bacterial counts can be measured.
  • the present inventors have conducted intensive studies on a rapid, simple, and highly sensitive measurement method of microorganisms captured on filtration membranes, and as a result, the bright spot ⁇ the background of background light emission appearing on these filtration membranes Found that the filtration membrane was contaminated with AMP and ATP. Furthermore, if this contamination is washed or removed by means such as electrophoresis or chemical or enzymatic modification or decomposition, noise luminescent spots and background luminescence will not appear, resulting in a more accurate and rapid bacterial count. They found that measurement was possible, and completed the present invention.
  • the present invention is selected from the group consisting of an organic solvent, sterilized water containing an organic solvent, sterilized water, an enzyme solution containing an adenate esterase, an acid, an alkali, a surfactant, and a bleaching agent.
  • the organic solvent include ethanol.
  • Bleaching agents include hypochlorous acid, hypobromite and hypoiodite, and salts thereof, such as sodium hypochlorite, potassium hypochlorite, calcium hypochlorite, barium hypochlorite, Methyl hypochlorite, Copper hypochlorite, Silver hypochlorite, Sodium hypobromite, Potassium hypobromite, Calcium hypobromite, Barium hypobromite, Methyl hypobromite, Next Copper hypobromite, silver hypobromite, sodium hypoiodite, potassium hypoiodite, calcium hypoiodite, barium hypoiodite, methyl hypoiodite, copper hypoiodite, and And silver iodate. Tween80 is mentioned as a surfactant. Also
  • Adenosine phosphates may be removed by combining a bleaching agent and a surfactant and bringing them into contact with a filtration membrane or a water absorption zone.
  • the bleaching agent may be hypochlorous acid, hypobromous acid or hypoiodic acid, or a salt thereof (eg, sodium hypochlorite, potassium hypochlorite, calcium hypochlorite, hypochlorous acid) Barium, methyl hypochlorite, copper hypochlorite, silver hypochlorite, sodium hypobromite, potassium hypobromite, calcium hypobromite, barium hypobromite, hypobromite Methyl, copper hypobromite, silver hypobromite, sodium hypoiodite, potassium hypoiodite, calcium hypoiodite, barium hypoiodite, methyl hypoiodite, hypoiodite Copper or silver hypoiodite) or a combination thereof is preferably combined using Tween80 as a surfactant.
  • the bleach at a concentration of 00005 to 5% with Tween 80 at a concentration of 0.0001 to 5%. It is particularly preferable to use sodium hypochlorite as a bleaching agent and Tween80 as a surfactant. In this case, the effective chlorine concentration is 0.00000 to 5
  • sodium hypochlorite at a concentration of% and Tween 80 at a concentration of 0.0001 to 5% are combined. It is particularly preferable to combine sodium hypochlorite having an effective chlorine concentration of 0.0005 to 0.5% with Tween80 having a 0.0001 to 5% concentration.
  • adenosine phosphates may be removed by bringing a bleaching agent into contact with a filtration membrane or a water absorption zone. In this case, it is preferable to use the above-mentioned type of bleaching agent, and it is more preferable to use a bleaching agent having a concentration of 0.0005 to 0.5%.
  • the above treatment of the present invention is preferably performed at a temperature of 10 to 150 ° C. During this treatment, it is also preferable to apply autoclaving.
  • the present invention is a filtration membrane or a water absorption zone treated by the above treatment method. Furthermore, the present invention is a method for measuring the number of bacteria in a sample, comprising the following steps.
  • the filtration membrane to be washed in the present invention is not particularly limited as long as it can capture microorganisms, and includes, for example, a commercially available hydrophilic or hydrophobic filtration membrane.
  • hydrophilic filtration membranes include hydrophilic polytetrafluoroethylene, hydrophilic polyvinylidene difluoride, hydrophilic polysulfone, hydrophilic polycarbonate, hydrophilic polyamide, hydrophilic polyethylene, hydrophilic polypropylene, etc.
  • a film-like or sheet-like material made of a hydrophilic plastic material or a material such as acetyl cellulose or nitrocellulose is used.
  • hydrophobic filtration membrane examples include, for example, PVDF (polyvinylidene difluoride), PTFE (polytetrafluoroethylene), PE (polyethylene), and the like, or a relatively large hydrophilic hydrophobic membrane such as PC (Polycarbonate), PP (polypropylene), PA (polyamide), PS (polysulfone) and the like can be used. Also, RMD Special Membrane Fill Yuichi (PVDF film with hydrophobic lattice) and polycarbonate film manufactured by Nippon Millipore are preferred. Next, the method for cleaning a filtration membrane of the present invention will be described.
  • examples of the solution for washing the filtration membrane include organic solvents such as methanol, ethanol, propanol, butanol, isopropanol, acetonitrile, acetone, and dimethyl sulfoxide, or sterilized water containing these organic solvents. Alternatively, only sterilized water may be used. Also optional It is possible to use sterile water in which the above-mentioned buffer or salt is dissolved, or to use a solution in which these are mixed with an organic solvent.
  • the concentration and type of these organic solvents are not particularly limited, but preferably 5 to 95% ethanol, particularly preferably 10 to 90% ethanol.
  • a surfactant may be further added to these cleaning liquids before use.
  • the surfactant include any one selected from Tween, SDS, SPAN, Triton, benzalkonium chloride, benzethonium chloride, and the like, and the concentration used is not particularly limited.
  • Tween80 is at least 0.0001% and at most 50%, more preferably at least 0.001% and at most 30%.
  • an acid or an alkali may be added to these cleaning liquids before use. Any acid or alkali can be used.
  • Any acid or alkali can be used.
  • the concentration to be used is 0.1% or more, preferably 1% or more.
  • the alkali ammonia, sodium hydroxide, potassium hydroxide and the like are preferable, and the concentration to be used is 0.1% or more, preferably 1% to 30%.
  • drying the filtration membrane it is preferable to put it in a petri dish to dry so that dust and the like do not adhere.
  • the drying temperature is not particularly limited, but preferably 40 ° C or more, particularly preferably 45 ° C or more. Appropriate.
  • the temperature at this time is also not particularly limited, but is preferably 30 ° C or more, and particularly preferably 40 ° C or more.
  • the adenosine phosphates of the filtration membrane can be treated by enzymatically or chemically modifying or decomposing them into substances not related to the reaction system.
  • the reagent to be used is not particularly limited, but is preferably an acid or an alkali, in addition to an enzyme such as adenosine phosphate deaminase which widely degrades adenosine phosphates.
  • acids hydrochloric acid or acetic acid is particularly preferred, and the concentration used is 0.1% or more, preferably
  • Potassium hydroxide and the like are preferable, and the concentration to be used is 0.1% or more, preferably 1% or more and 30% or less.
  • the temperature during the reaction is not particularly limited, but is 40 ° C or more, especially
  • 60 ° C. or higher is preferred. It is also particularly preferable to carry out autoclave treatment at a temperature of 100 ° C or higher (for example, 121 ° C).
  • adenosine phosphate esters in the filtration membrane may be decomposed using a bleaching agent or the like.
  • Bleaching agents include oxidizing bleaches and reducing bleaches.
  • Oxidative bleaching agents include peroxides and chlorine compounds. Examples of the peroxides include hydrogen peroxide, sodium peroxide, sodium perborate, potassium permanganate, and the like.
  • Examples of the chlorine compound include salad powder, sodium hypochlorite, sodium chlorite and the like.
  • the reducing bleaching agent include iodide, sodium bisulfite, hydrosulfite, and sodium dithionite.
  • potassium bisulfite, sodium hyposulfite, potassium pyrosulfite, nitrogen dioxide, benzoyl peroxide, ammonium persulfate, chlorine dioxide and the like can be used.
  • the bleaching agent include hypochlorous acid, hypobromite, hypoiodic acid, and salts thereof, such as sodium hypochlorite, potassium hypochlorite, calcium hypochlorite, and hypochlorous acid.
  • a bleaching agent having a concentration of 0.0005 to 0.5%.
  • an appropriate surfactant may be used in combination.
  • the bleaching agent may be hypochlorous acid, hypobromite, hypoiodic acid, or a salt thereof, such as sodium hypochlorite, potassium hypochlorite, calcium hypochlorite, hypochlorous acid.
  • sodium hypochlorite and Tween80 in combination, and it is preferable to use sodium hypochlorite having an effective chlorine concentration of 0.00000 to 5% and Tween80 at a concentration of 0.0001 to 5%. .
  • a bleaching agent and a surfactant are used in combination, it is preferable to use a bleaching agent having a concentration of 0.0005 to 0.5% and a surfactant having a concentration of 0.0001 to 5%.
  • the temperature during the reaction is arbitrary, but is preferably iOt or more, preferably 30: or more, particularly preferably 40 ° C. or more and 150 or less. In this treatment, autoclaving is also preferable.
  • an enzyme for example, adenosine phosphate deaminase solution, or adenosine phosphate deaminase and another enzyme (eg, avirase, alkaline phosphatase, acid phosphatase, hexokinase or adenosine triphosphatase, or a combination thereof)
  • a mixed solution or the like can be used.
  • the final concentration of adenosine phosphate deaminase is at least 0.001 l / ml, preferably at least 0.01 U / ml and at most 10 U / ml.
  • the pH during the reaction is preferably from pH 4.5 to pH 8.5, particularly preferably from pH 5 to pH 8.
  • the temperature during the reaction is preferably from 25 to 60 ° C, more preferably from 30 to 55 ° C.
  • liquid-contacting parts such as the water absorption zone of the sprayer used for measurement, that is, the parts that come into contact with the luminescent reagent and the extraction reagent are contaminated with adenosine phosphates. May cause ground emission. Therefore, it is necessary to remove adenosine phosphates by an enzyme treatment or the like in advance.
  • a water absorption zone examples thereof include those used for Matsushita Electric Works ultrasonic sprayer (EW622) and those used for automatic sprayer manufactured by Nippon Millipore.
  • the enzyme to be used is, for example, adenosine phosphate deaminase solution or adenosine phosphate deaminase and other enzymes (eg, apyrase, alkaline phosphatase, acid phosphatase, hexokinase, adenosine triphosphatase).
  • a mixed solution or the like can be used.
  • the final concentration of adenosine phosphate kinase is 0.001 U / ml or more, preferably 0.01 U / ml or more and 10 U / ml or less.
  • the pH during the reaction is preferably from pH 4.5 to pH 8.5, and particularly preferably from pH 5 to pH 8.
  • the temperature during the reaction is preferably 25 ° C or higher and 60 ° C or lower, but is more preferably 30 ° C or higher and 55 ° C or lower.
  • drying the water absorption zone after the above treatment air drying, dry heat, or the like can be used.
  • the drying temperature is not particularly limited, but preferably 40 ° C or more, particularly preferably 45 ° C or more. Appropriate.
  • a method of drying by blowing a clean wind through a HEPA filter or the like is also preferable.
  • the temperature at this time is also not particularly limited, but is preferably 30 ° C or more, and particularly preferably 40 ° C or more.
  • the microorganism according to the present invention means yeasts, molds, mushrooms, bacteria, actinomycetes, unicellular algae, viruses, protozoa, animals or non-differentiated cells and tissue cultures.
  • the sample liquid is not particularly limited, but examples include the following.
  • Beverages peel, wine, juice, etc.
  • Body fluid urine, feces, blood, sputum, pus, etc.
  • a solution in which the above sample solution is suspended in an appropriate solvent may be used as the sample solution.
  • an appropriate solvent for example, distilled water, physiological saline, phosphate buffer solution, Tris buffer solution, sodium acetate buffer solution, etc.
  • the sample solution contains a solid content
  • the sample solution is suspended in an appropriate solvent (eg, distilled water, physiological saline, phosphate buffer, Tris buffer, acetate buffer, etc.) or a mixer.
  • a solution that has been homogenized by, for example, and used as a solution can be used.
  • the means for capturing the microorganisms on the filtration membrane is not particularly limited, but, for example, is by adding a sample solution which seems to contain microorganisms to the filtration membrane. In the present invention
  • Adding the sample solution to the filtration membrane means applying the sample solution to the filtration membrane, specifically, filtering the sample solution using the filtration membrane, and spotting the sample solution on the filtration membrane.
  • a filter membrane after washing by the above method is attached to a cup-shaped filtration container (for example, Milliflex filter unit manufactured by Nippon Millipore Co., Ltd., Sterifil), and the sample solution is suction-filtered
  • a cup-shaped filtration container for example, Milliflex filter unit manufactured by Nippon Millipore Co., Ltd., Sterifil
  • a method of capturing microorganisms on a filtration membrane is employed. After the filtration is completed, remove the filtration membrane from the filter, and if necessary, wash and air-dry the filtration membrane.
  • an isotonic solution such as a physiological saline solution for washing the filtration membrane in order to prevent cell rupture.
  • the filter membrane After filtering the sample, the filter membrane may be placed on an agar medium containing adenosine phosphate deminase, cultured for an appropriate time, dried, and sprayed with an extraction reagent. As a result, the amount of luminescence from bacteria can be increased.
  • ATP scavenger a reagent containing ATP-degrading enzyme
  • background AT ATP other than ATP contained in the microbial cells
  • Treating the filtration membrane with an ATP scavenger reduces background luminescence in the bioluminescence reaction and increases the sensitivity of microbial measurement. Is improved.
  • ATP scavenger examples include adenosine phosphate deaminase solution, adenosine phosphate deaminase and other enzymes (eg, apyrase, alkaline phosphatase, acid phosphatase, hexokinase or adenosine triphosphase, or a combination thereof). And the like can be used.
  • the final concentration of adenosine phosphate kinase in the ATP scavenger is 0.001 U / ml or more, and preferably 0.01 to 10 U / ml.
  • the ATP scavenger When the filter membrane is brought into contact with the ATP scavenger, the ATP scavenger may be dropped or sprayed on the filter membrane or the filter membrane may be immersed in the ATP scavenger.
  • the ATP scavenger After erasing the backbisd ATP, it is desirable to remove or deactivate the ATP scavenger on the filtration membrane.
  • the removal of the ATP scavenger is carried out by washing the filter membrane with ATP-depleted ultrapure water or a buffer solution. Further, the ATP scavenger can be deactivated by acting an inhibitor against the scavenger. .
  • coformycin can be used as an inhibitor of the scavenger.
  • Coformycin is known as an inhibitor of adenosine phosphate deaminase (THE JOURNAL OF ANTIBI0TI CS, SE. A Vol. 20 No. 4 227-231).
  • the inhibitor When an inhibitor of the ATP scavenger is allowed to act, the inhibitor may be dropped or sprayed onto the filtration membrane. More preferably, the inhibitor may be added to the ATP extractant described below and added or sprayed onto the filtration membrane. Next, the biological components in the microorganisms captured on the filtration membrane are extracted.
  • the method for extracting the biological component is not particularly limited.
  • a known ATP extractant may be dropped or sprayed on the filtration membrane.
  • ATP extractants include, for example, a mixture of ethanol and ammonia
  • Methanol, ethanol, surfactants benzetonium chloride, benzalcodium chloride, Triton X100, etc.
  • trichloroacetic acid perchloric acid, etc.
  • a mixed solution of ethanol and ammonia is suitable as an ATP extractant because it is easy to volatilize after the ATP extraction operation and inhibits the activity of the ATPase.
  • the ATP extractant is sprayed using, for example, an ultrasonic sprayer (Matsushita Electric Works) for 5 seconds to 5 seconds. It may be done for about a minute. After ATP extraction, if necessary, the filtration membrane is dried to remove the ATP extractant.
  • an ultrasonic sprayer Matsushita Electric Works
  • the biological component in the microorganism according to the present invention means a substance contained in the microorganism and involved in the ATP conversion reaction or the bioluminescence reaction of the present invention.
  • Such substances include, for example, adenosine phosphate (ATP, AMP, ADP, cyclic AMP, RNA, etc.), PPDK, phosphoenolpyruvate, pyrophosphate, luciferin, luciferase, pyruvate kinase, acetate acetate Creatine kinase, or cyclic 3 ', 5'-nucleotide phosphodiesterase.
  • the biological component of the present invention substances involved in the production of the above-mentioned biological components are also included in the biological component of the present invention.
  • the ATP conversion reagent and the bioluminescent reagent are dropped or sprayed on the filtration membrane from which the biological components in the microorganisms have been extracted.
  • the ATP conversion reaction reagent referred to in the present invention is a reagent containing a substance involved in the ATP conversion reaction, for example, pyruvate orthophosphate dikinase (hereinafter, “PPDK”).
  • PPDK pyruvate orthophosphate dikinase
  • the ATP conversion reaction referred to in the present invention refers to any reaction system in which ATP is generated during the reaction, and includes, for example, a reaction system in which AMP is directly generated from AMP.
  • One of the substances that contribute to the ATP conversion reaction is, for example, an enzyme that catalyzes the ATP conversion reaction.
  • Enzymes that catalyze this ATP conversion reaction include, for example, pyruvate orthophosphate dikinase (PPDK) and phosphoenol pyruvate synthetase.
  • the following ATP conversion reaction can be used by using a substance involved in the ATP conversion reaction.
  • ATP is generated by combining AMP phosphotransferase and adenylate kinase (Applied and Environmental Microbiology, May
  • a ATP is produced from AMP using a combination of AMP-PolyP phosphotransferase (AMP-Polyphosphate kinase) and Polyphosphate kinase (Polyphosphate kinase).
  • AMP-Polyphosphate kinase AMP-Polyphosphate kinase
  • Polyphosphate kinase Polyphosphate kinase
  • the ATP conversion reaction reagent in the present invention is, for example, a reagent containing the following substances (a) to (2).
  • PPDK is a known enzyme that catalyzes the reaction of producing ATP, pyruvate and phosphoric acid by acting on AMP, phosphoenorubyruvic acid and pyrophosphate in the presence of magnesium ion, and also catalyzes the reverse reaction.
  • ATP is generated by the reaction between the above reagent and AMP in the reaction system.
  • the AMP in the reaction system as referred to herein is that contained as a biological component in the microorganism and that generated with the consumption of ATP in the bioluminescence reaction.
  • ATP conversion reaction reagent containing the substances (a) to (2) may be added to the ATP conversion reaction reagent containing the substances (a) to (2).
  • substances (a) to (2) For example, when cyclic 3 ', 5'-nucleotide phosphodiesterase is added, ATP is generated not only from AMP but also from cyclic AMP.
  • ATP is generated not only from AMP but also from ADP.
  • a reagent which is a combination of the above-mentioned substances (a) to (2) and another substance and which contains a substance which participates in the reaction that generates ATP during the reaction is an ATP conversion reaction reagent of the present invention.
  • the physicochemical properties and production method of PPDK which is one of the substances involved in the ATP conversion reaction, are known, and their availability is relatively easy.
  • Examples of plant-derived PPDK include enzymes derived from corn leaves [Biochemis try 12, 2862-2867 (1973)] and sugarcane leaves [The Biochemical Journal 114, 117-125 (1969)].
  • Microbial sources include, for example, Propionibacterium shermanii (Biochemistry 10, 721-729 (1971)), Acetobacter xylinum (Ace tobacter xyl inum) [Journal of Bacteriology (1970)], Bacteroides symbiosus [Me thods in Enzymolog y 42, 199-212 (1975)] and the genus Microbispora [eg Microbispora thermorosea IF0 14047] and the like.
  • a natural PPDK purified from the above organism can be used.
  • a mutant PPDK in which one or more (eg, one or several) amino acids in the amino acid sequence of the natural PPDK are introduced, deleted, or substituted with a mutation within a range not to lose the enzyme activity can also be used.
  • Natural PPDK can also be obtained by genetic engineering techniques. That is, the native PPDK gene may be introduced into an appropriate vector-one host system, and PPDK may be collected from the obtained culture of the recombinant microorganism. In addition, the PPDK gene can be obtained from maize (J. Biol. Chem. 263, 11080-11083 (1988)), Flaveria trinervia (FEBS Let t. 273, 116-121 (1990)), etc. It has already been cloned from living things.
  • the PPDK can be obtained from a mutant of the above organism.
  • mutant PPM can be obtained by genetic engineering techniques. That is, it is added to one or more (eg, one or several) bases in the base sequence of the natural PPDK gene.
  • a mutant PPDK gene into which mutations such as deletion, substitution, etc. have been introduced is prepared and introduced into an appropriate vector-host system, and a mutant PPDK gene is collected from a culture of the obtained recombinant microorganism. I just need.
  • Methods for introducing a mutation into a gene include, for example, a natural PPDK gene and a mutagenic agent (hydroxylamine, nitrite, sulfurous acid, 5-promouracil, etc.)
  • a site-specific mutagenesis method using a PCR method can be widely used. Furthermore, It is also possible to construct a mutant PPDK gene having a mutation at a desired site by annealing the chemically synthesized DNA.
  • the bioluminescent reagent in the present invention is not particularly limited as long as it is a reagent containing a substance involved in a bioluminescent reaction.
  • the bioluminescent reagent of the present invention is, for example, a reagent containing a substance involved in a luciferin-luciferase luminescence reaction, and specifically, for example, a reagent containing the following substances (a) to (c) It is.
  • a luminescence reaction occurs when the above reagent reacts with ATP in the reaction system. At that time, ATP is consumed and AMP is generated.
  • the ATP in the reaction system as referred to herein includes those contained as biological components in microorganisms and those produced by the ATP conversion reaction.
  • luciferin and luciferase those derived from, for example, insects (Genji firefly, Heike firefly, North American firefly, Hikarimemushimushi, Tsubotaru) can be used.
  • Luciferase is added to or deleted from one or more amino acids in the amino acid sequence of natural luciferase purified from the luminescent tissue of the above organism, natural luciferase prepared by genetic engineering techniques, or natural luciferase. Mutant luciferase into which a mutation such as loss or substitution has been introduced can be used.
  • AMP is generated from RNA by adding liponuclease (RNase) to the reaction system, and the AMP becomes ATP in the ATP conversion reaction.
  • RNase liponuclease
  • a biological component involved in the bioluminescence reaction is mainly ATP, and there is a problem that the amount of luminescence is attenuated with consumption of the ATP.
  • ATP is newly generated from various adenosine phosphates (AMP, cyclic AMP, ADP, RNA, etc.) and consumed in the bioluminescence reaction. ATP is also played. As a result, the luminescence amount and the luminescence time are increased, so that microorganisms can be measured quickly, easily and with high sensitivity.
  • the conditions of the ATP conversion reaction and the bioluminescence reaction are not particularly limited. What is necessary is just to set suitably. That is, the ATP conversion reaction reagent and the bioluminescent reagent may be used as a mixture, or may be sequentially added to the filtration membrane.
  • a mixture of the ATP conversion reaction reagent and the bioluminescent reagent it is preferable to use a mixture of the ATP conversion reaction reagent and the bioluminescent reagent.
  • a mixed solution having the following composition can be used.
  • Luciferase (F) Luciferase; final concentration 0.1 mg / ml or more, preferably 0.5 to 20 mg / nil
  • ATP is regenerated from AMP generated by consumption of ATP in the bioluminescence reaction, so that the luminescence amount and the luminescence time are increased.
  • ammonium sulfate, dithiothreitol (DTT), EDTA, HEPES or Tricine is a substance that is preferably added to improve the stability or buffer capacity of the solution.
  • DTT dithiothreitol
  • EDTA EDTA
  • HEPES Tricine
  • Tricine is a substance that is preferably added to improve the stability or buffer capacity of the solution.
  • perserum albumin, sucrose and the like can be used.
  • cyclic AMP which is a biological component in a microorganism
  • cyclic 3 ′, 5′-nucleotide phosphodiesterase (0.01 ⁇ l / ml or more, preferably 0.02 to 10 l / ml).
  • the ATP conversion reagent and bioluminescent reagent contain adenosine lysate, an ATPase. Acid deaminase may be added. If ATP is contaminated in the above reagent, ATP in the reagent will be eliminated by the addition of adenosine phosphate kinase. In that case, it is preferable to add coformycin to the ATP extractant. In this way, when the above reagent is added to the filtration membrane, decomposition of ATP derived from microorganisms can be prevented.
  • adenosine phosphate deaminase When adding adenosine phosphate deaminase to the above mixed solution of the ATP conversion reaction reagent and the bioluminescent reagent, add it so that the concentration is 0.01 l / ml or more, preferably 0.05 to 10 U / ml. do it.
  • the amount of luminescence generated on the filtration membrane can be measured by a luminometer, for example, a luminescence reader BLR-201 (improved type) manufactured by Aroka.
  • the number of viable bacteria is measured by imaging the bright spots on the filtration membrane using a bioluminescence image analysis system device, for example, ARGUS-50 / CL (with taper fiber: manufactured by Hamamatsu Photonics KK). It is also possible.
  • a PPM luminescence reagent (a mixed reagent of an ATP conversion reaction reagent and a bioluminescence reagent) was used as the reagent of the present invention.
  • HSLU bioluminescent reagent not containing ATP conversion reagent
  • DPPDK from Microbispora thermorosea IF014047
  • lOOmM sodium acetate buffer containing lmM EDTA
  • pH 5.0 pH 5.0
  • the amount of enzyme whose 0D value changes by 2.4 per minute is defined as 1 unit (U).
  • the amount of enzyme that releases 1.0 Omol of inorganic phosphoric acid per minute from ATP is defined as 1 unit (U).
  • a filtration membrane RMD special membrane filter manufactured by Nippon Millipore Co., Ltd. (with 0.45 ⁇ m hydrophobic lattice, diameter 47mm) was immersed in 70% ethanol. This is set in a filter equipped with a polypropylene pre-filter (0.6 m), manufactured by Nippon Millipore Co., Ltd. and washed with 50 ml of 70% ethanol, and washed with 100 ml of 70% ethanol in advance. did. Thereafter, the membrane was washed twice with 100 ml of 20% ethanol, the membrane filter was removed from the filter, turned over, and set again in the filter.
  • the membrane was washed with 30 ml of ultrapure water, removed from the filter, placed in a sterile petri dish, and dried at 50 ° C for 15 hours.
  • the PPM luminescent reagent was applied to the membrane before washing by an ultrasonic sprayer manufactured by Matsushita Electric Works (EW622). After setting in a RMDS device manufactured by Millipore Japan, luminescence was integrated for 5 minutes and measured at bit 0-4. The washed membrane was treated in the same manner and compared.
  • a filtration membrane RMD special membrane filter manufactured by Nippon Millipore Co., Ltd. (0.45 m with hydrophobic grid, diameter 47 mm) was immersed in 70% ethanol. Then, it was placed in 200 ml of 10% acetic acid and placed in a 200 ml pressure bottle and capped. This was autoclaved at 121 ° C for 200 minutes. After the temperature dropped, the membrane was taken out and rinsed three times with ultrapure water to remove excess acetic acid.
  • the membrane before the treatment was sprayed with a PPM luminescent reagent using an ultrasonic atomizer (EW622) manufactured by Matsushita Electric Works, and set on a RMDS device manufactured by Millipore Japan. After that, the luminescence was integrated for 5 minutes and measured at bit 0-4. The treated film was similarly treated and compared.
  • EW622 ultrasonic atomizer
  • RMD special membrane filter 1 manufactured by Nippon Millipore, 0.45 Mm with hydrophobic lattice, diameter 471M1 as a filtration membrane was immersed in 70% ethanol. Thereafter, the filter was set on a filter, and 1 Oml of a 10 mM HEPES buffer solution (pH 7.0) containing 1.ml/ml of adenosine phosphate deaminase was passed through to wash out excess 70% ethanol.
  • the plate was immersed in 20 ml of lOmM HEPES buffer (pH 7.0) containing 1.0 U / ml adenosine phosphate deaminase in a sterile petri dish and treated at 50 ° C for 6 hours. After the treatment, only the filter membrane was transferred into a new sterile petri dish and dried at 50 ° C overnight.
  • lOmM HEPES buffer pH 7.0
  • the PPDK luminescence reagent was sprayed on the membrane before the treatment with an ultrasonic sprayer (EW622) manufactured by Matsushita Electric Works, and set on a RMDS device manufactured by Millipore Japan. After that, the luminescence was integrated for 5 minutes and measured at bit 0-4. The treated film was similarly treated and compared.
  • EW622 ultrasonic sprayer manufactured by Matsushita Electric Works
  • the film before the treatment emitted light over the entire surface, and 20 noise luminescent spots were observed.
  • the film after the treatment showed no noise luminescent spot and no background light emission.
  • Nippon Millipore RMD Special Membrane Filter (0.45 m hydrophobic) 100 pieces with a lattice and 47 imn in diameter were immersed in 70% ethanol. Then, the mixture was put in a 200 ml mixed solution of 0.0005% sodium hypochlorite and 0.01% Tween80 with an effective chlorine concentration of 200 ml and sealed in a 200 ml pressure-resistant bottle. This was treated at 60 ° C for 5 hours. After the treatment, the filtration membrane was taken out and rinsed sufficiently with ultrapure water to remove excess sodium hypochlorite.
  • the PPDK luminescence reagent was sprayed on the membrane before the treatment with an ultrasonic sprayer (EW622) manufactured by Matsushita Electric Works, and set on a RMDS device manufactured by Millipore Japan. After that, the luminescence was integrated for 5 minutes and measured at bit 0-4. The treated film was similarly treated and compared.
  • EW622 ultrasonic sprayer manufactured by Matsushita Electric Works
  • the film before the treatment emitted light from the entire surface, but the film after the treatment did not show any noise luminescent spot or background light emission.
  • This method was excellent in that a large number of films could be processed at once.
  • the PPDK luminescence reagent was sprayed on the membrane before the treatment with an ultrasonic sprayer (EW622) manufactured by Matsushita Electric Works, and set on a RMDS device manufactured by Millipore Japan. After that, the luminescence was integrated for 5 minutes and measured at bit 0-4. The treated film was similarly treated and compared.
  • EW622 ultrasonic sprayer manufactured by Matsushita Electric Works
  • the film before the treatment emitted light from the entire surface, but the film after the treatment did not show any noise luminescent spot or background light emission.
  • This method was excellent in that a large number of films could be processed at once.
  • Tween80 mixture was placed in 200 ml and placed in a 200 ml pressure bottle and capped. This was treated at 60 ° C for 6 hours. After treatment, remove the filtration membrane, rinse thoroughly with ultrapure water and remove excess Potassium hypochlorite was removed.
  • the PPDK luminescence reagent was sprayed on the membrane before the treatment with an ultrasonic sprayer (EW622) manufactured by Matsushita Electric Works, and set on a RMDS device manufactured by Millipore Japan. After that, the luminescence was integrated for 5 minutes and measured at bit 0-4. The treated film was similarly treated and compared.
  • EW622 ultrasonic sprayer manufactured by Matsushita Electric Works
  • the film before the treatment emitted light from the entire surface, but the film after the treatment did not show any noise luminescent spot or background light emission.
  • This method was excellent in that a large number of films could be processed at once.
  • Nippon Millipore RMD Special Membrane Filter (0.45 m with hydrophobic lattice, diameter 47 mm) as filter membrane 100 pieces of effective chlorine concentration 0.05% sodium hypochlorite 200 ml and put in 200 ml pressure bottle I closed the lid. This was treated at room temperature for 6 hours. After the treatment, the filtration membrane was taken out and rinsed sufficiently with ultrapure water to remove excess sodium hypochlorite.
  • the membrane before the treatment was sprayed with a PPM luminescent reagent using an ultrasonic atomizer (EW622) manufactured by Matsushita Electric Works, and set on a RMDS device manufactured by Millipore Japan. After that, the luminescence was integrated for 5 minutes and measured at bit 0-4. The treated film was similarly treated and compared.
  • EW622 ultrasonic atomizer
  • the film before the treatment emitted light from the entire surface, but the film after the treatment did not show any noise luminescent spot or background light emission.
  • This method was excellent in that a large number of films could be processed at once.
  • Nihon Millipore RMD Special Membrane Filter (0.45 Mm with hydrophobic lattice, diameter 47 mm) is a filtration membrane. Effective chlorine concentration 0.005% sodium hypochlorite, 0.01% TweenSO mixed solution Put in 200ml, put in a 200ml pressure bottle and capped. This was treated at room temperature for 4 hours. After the treatment, the filtration membrane was taken out and rinsed sufficiently with ultrapure water to remove excess sodium hypochlorite.
  • the membrane before the treatment was sprayed with a PPM luminescent reagent using an ultrasonic atomizer (EW622) manufactured by Matsushita Electric Works, and set on a RMDS device manufactured by Millipore Japan. After that, the light emission is integrated for 5 minutes and bit 0-4 Measured.
  • the treated film was similarly treated and compared.
  • the film before the treatment emitted light from the entire surface, but the film after the treatment did not show any noise luminescent spot or background light emission.
  • This method was excellent in that a large number of films could be processed at once.
  • Nippon Millipore RMD Special Membrane Filter (0.45 / m with hydrophobic lattice, diameter 47mm), which is a filtration membrane, put 200 sheets of effective chlorine concentration 0.005% sodium hypochlorite in 200ml and put it in a 200ml pressure bottle I put it in and closed it. This was treated at room temperature for 15 hours. After the treatment, the filtration membrane was taken out and rinsed sufficiently with ultrapure water to remove excess sodium hypochlorite.
  • the PPDK luminescence reagent was sprayed on the membrane before the treatment with an ultrasonic sprayer (EW622) manufactured by Matsushita Electric Works, and set on a RMDS device manufactured by Millipore Japan. Thereafter, luminescence was integrated for 5 minutes and measured at Mt 0-4. The treated film was similarly treated and compared.
  • the film before the treatment emitted light from the entire surface, but the film after the treatment did not show any noise luminescent spot or background light emission.
  • This method was excellent in that a large number of films could be processed at once.
  • the water absorption zone for an ultrasonic atomizer (EW622) manufactured by Matsushita Electric Works was immersed in sterilized ultrapure water containing 0.1 ⁇ g / ml of adenosine phosphate deaminase, and treated with 3rc for 1 hour. This was placed in a sterile petri dish and dried at 50 ° C for 15 hours. This was immersed in 2 ml of ultrapure water and stirred vigorously. The obtained solution 5a1 was added to PPDK luminescence reagent 1001, and the measurement was performed using a Lumitester C-100. The measured value was 520 RLU.
  • Example 11 A filtration membrane RMD special membrane filter manufactured by Nippon Millipore Co. (0.45 / 1 m with hydrophobic grid, diameter 47 mni) was immersed in 5% Tween80. Set this in a filter equipped with a polypropylene pre-filter (0.6 / xm) and MF mesh filter AP32, manufactured by Nippon Millipore Co., Ltd., previously washed with 5% Tween80 and 100 ml, and made up to 100 ml with 5% Tween80. After washing twice, the membrane filter was removed from the filter, turned over, and set back in the filter. Further, it was washed with 30 ml of ultrapure water to remove excess Tween80, removed from the filter, placed in a sterile petri dish, and dried at 50 ° C for 15 hours.
  • the membrane before washing was sprayed with a PPDK luminescent reagent using an ultrasonic atomizer (EW622) manufactured by Matsushita Electric Works, and set on a RMDS device manufactured by Millipore Japan. After that, the luminescence was integrated for 5 minutes and measured at bit 0-4. The washed membrane was treated in the same manner and compared.
  • EW622 ultrasonic atomizer
  • the film before the treatment emitted light on the entire surface, but the film after the treatment did not show any noise luminescent spots or background emission.
  • the membrane before washing was sprayed with a PPDK luminescent reagent using an ultrasonic atomizer (EW622) manufactured by Matsushita Electric Works, and set on a RMDS device manufactured by Millipore Japan. After that, the luminescence was integrated for 5 minutes and measured at bit 0-4. The washed membrane was treated in the same manner and compared.
  • EW622 ultrasonic atomizer
  • the film before cleaning emitted light over the entire surface, and 24 noise luminescent spots were observed.
  • the film after cleaning showed no noise luminescent spot and no background light emission.
  • Nippon Millipore RMD special membrane filter (0.45 m hydrophobic) A grid with a diameter of 47 mm) was immersed in 70% ethanol. This was set in a filter equipped with Nippon Millipore's polypropylene prefill Yuichi (0.6 m) and MF mesh spacer AP32 previously washed with 100% 70% ethanol and 50 ml of 70% ethanol. And washed. After that, the membrane was washed twice with 100 ml of 20% ethanol, the membrane filter was removed from the filter, turned over, and set again in the filter. Further, the membrane was washed with 30 ml of ultrapure water, removed from the filter, placed in a sterile petri dish, and dried at 50 ° C for 15 hours.
  • HSLU was sprayed on the membrane before washing with an ultrasonic atomizer (EW622, manufactured by Matsushita Electric Works). Then, it was set on a RMDS device manufactured by Japan Millipore. After that, the luminescence was integrated for 5 minutes and measured at bit 0-4. The washed membrane was treated in the same manner and compared.
  • the membrane filter prepared in Example 5 was set on a filter, and washed twice with 20 ml of ultrapure water. Then, add 10 ml of the washing solution (0.05% glucose, 0.05% fructose, 1 mM HEPES pH 7.0), and add the bacterial dilution (5% sucrose, 0.05% glucose, 0.05% fructose, 10% HEPES, 10 mM HEPES). Escherichia coli ATCC25922 appropriately diluted with pH 7.0) was added. Next, the plate was washed twice with 10 ml of a washing solution, and then washed with 10 ml of a washing solution containing 0.1 mg / ml adenosine phosphate deaminase.
  • the washing solution 0.05% glucose, 0.05% fructose, 1 mM HEPES pH 7.0
  • the bacterial dilution 5% sucrose, 0.05% glucose, 0.05% fructose, 10% HEPES, 10 mM
  • the membrane was removed from the filter, set in a holder, placed in a petri dish, opened slightly, and air-dried. After drying, the ATP extractant was sprayed with a Matsushita Electric Works ultrasonic sprayer (EW622) for 5 seconds and air-dried. Thereafter, a PPDK luminescence reagent was sprayed for 5 seconds with an ultrasonic atomizer (EW622) manufactured by Matsushita Electric Works, and set on an RMDS device to integrate the luminescence amount for 5 minutes. The number of appearing bright spots was measured with bitO-4. On the other hand, the number of bacteria (Colony Forming Unit) (CFU) measured by pour culture on a standard agar medium was measured and shown in Table 1.
  • CFU Coldy Forming Unit
  • the present invention (piece) pour culture method (GFU)
  • the membrane was removed from the filter, set in a holder, placed in a petri dish, opened slightly, and air-dried. After drying, the ATP extractant was sprayed with a Matsushita Electric Works ultrasonic sprayer (EW622) for 5 seconds and air-dried. Then, the PPDK luminescent reagent was sprayed for 5 seconds with an ultrasonic atomizer (EW622) manufactured by Matsushita Electric Works, set on an RMDS device, and the luminescence was integrated for 5 minutes. The number of bright spots was measured using MtO-4. On the other hand, the number of bacteria (CFU) measured by pour culture on a standard agar medium was measured and shown in Table 2.
  • a method for washing a filtration membrane, and a method for measuring the number of bacteria using the washed filtration membrane are provided.

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Abstract

La présente invention concerne un procédé pour traiter une membrane filtrante ou une zone absorbant l'eau. Ce procédé consiste à mettre en contact ladite membrane ou ladite zone avec au moins un élément choisi dans le groupe formé par un solvant organique, de l'eau stérilisée contenant un solvant organique, de l'eau stérilisée, une solution enzymatique contenant une adénosine phosphatase, un acide, un alcali, un agent tensio-actif et un agent de blanchiment, afin d'éliminer l'adénosine phosphate qui adhère à la membrane filtrante ou à la zone absorbant l'eau.
PCT/JP2002/004733 2001-05-16 2002-05-16 Procede pour traiter une membrane filtrante, membrane filtrante et procede pour compter rapidement un nombre de micro-organismes a l'aide de cette membrane filtrante WO2002092202A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105617869A (zh) * 2014-11-07 2016-06-01 珠海格力电器股份有限公司 一种超滤膜滤芯的简易清洗方法
JPWO2017170973A1 (ja) * 2016-03-30 2019-02-07 東レ株式会社 膜モジュールによる微生物培養液のろ過方法

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS61209007A (ja) * 1985-03-12 1986-09-17 Nitto Electric Ind Co Ltd 補酵素の選択的分離方法
JPH10309446A (ja) * 1997-03-13 1998-11-24 Nok Corp 浄化装置
JPH1169994A (ja) * 1997-06-17 1999-03-16 Kikkoman Corp 微生物の測定法
JP2000189197A (ja) * 1998-12-25 2000-07-11 Kikkoman Corp Ａｔｐ消去剤、ａｔｐ消去法および細胞内ａｔｐの測定法

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS61209007A (ja) * 1985-03-12 1986-09-17 Nitto Electric Ind Co Ltd 補酵素の選択的分離方法
JPH10309446A (ja) * 1997-03-13 1998-11-24 Nok Corp 浄化装置
JPH1169994A (ja) * 1997-06-17 1999-03-16 Kikkoman Corp 微生物の測定法
JP2000189197A (ja) * 1998-12-25 2000-07-11 Kikkoman Corp Ａｔｐ消去剤、ａｔｐ消去法および細胞内ａｔｐの測定法

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105617869A (zh) * 2014-11-07 2016-06-01 珠海格力电器股份有限公司 一种超滤膜滤芯的简易清洗方法
JPWO2017170973A1 (ja) * 2016-03-30 2019-02-07 東レ株式会社 膜モジュールによる微生物培養液のろ過方法
EP3438243A4 (fr) * 2016-03-30 2019-11-06 Toray Industries, Inc. Procédé de filtration d'une solution de culture microbienne à l'aide d'un module à membrane

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