JP2009247331A - Method for detecting microorganism and apparatus for measuring the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To accurately detect and measure microorganisms by efficiently retrieving microorganisms in soybean milk. <P>SOLUTION: In preparing a sample, a reagent A is added to soybean milk and agitated to react the reagent A with soybean milk. Subsequently a reagent B is added and agitated to carry out a reaction. Further, a reagent C is added, agitated and incubated to finish the sample preparation. In staining the specimen, the total amount of the liquid after the sample preparation is filtered through a microorganism-retrieving filter 7 and residue components other than the microorganisms on the surface of the filter are washed with filter-sterilized water. A reagent D for staining the microorganisms is dripped on the filter 7 and spread over the whole surface of filter 7 to carry out a staining, after finishing the staining, an excess of the reagent D is washed off to complete the staining of the specimen to be measured. In measuring the specimen, the filter 7 for retrieving the microorganisms is set on the testing table 6 of a measuring device to measure the microorganisms, and the measuring operation is completed. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a microorganism detecting method and a microorganism measuring device for performing a microorganism test in soymilk.

  Conventionally, this type of microbiological testing method is a smear agar culture method in which 0.1 ml of a sample is dropped on an agar medium containing nutrient components and smeared with a congeal stick, or a pour which pours 1 ml of a sample without the agar medium solidifying. A culture method is used in which microorganisms are cultured by a culture method, a liquid culture method in which 0.1 ml or 1 ml of a specimen is added in a liquid medium, and the proliferation activity is detected. The culture method test is currently the most widely used means for detecting microorganisms. However, it takes 24 to 48 hours for culturing and cultivates microorganisms that do not match the culture conditions (temperature, time, medium nutrients). Issues that cannot be mentioned are issues.

As a method for measuring microorganisms using a membrane filter, a solution containing adenosine triphosphate (hereinafter referred to as ATP) -degrading enzyme is applied to the membrane filter, and then the microorganisms in the specimen are filtered and collected on the dried membrane filter. After collecting and culturing for a required time if necessary, luciferin and luciferase react with ATP by spraying a liquid extraction reagent for extracting ATP and a luminescence reagent luciferin luciferase in the form of a mist. One photon was emitted by oxidation of the molecule luciferin, and the emission was captured with a high-sensitivity CCD camera to measure microorganisms (see, for example, Patent Document 1).
Japanese National Patent Publication No. 9-512713

  Such a conventional culture method, which is a microbiological test method, requires 24-48 hours or more of culture time until a test result is obtained, and waits for the test result at the stage of production of fresh food or product shipment. This is a major disadvantage in terms of time and economics. In some cases, there is a problem that the product must be shipped before the test results are known, and the time required to measure the number of microorganisms in the sample is reduced. A short time is required.

  In addition, in the inspection of specimens that contain fluorescent foreign substances that emit light similar to microorganisms in the specimen, a clear difference between fluorescent foreign substances that emit light similar to microorganisms and the fluorescence of microorganisms cannot be obtained. There is a problem that the number of microorganisms cannot be measured, and it is required to accurately measure the number of microorganisms.

  The present invention solves the conventional problems, shortens the time required for measuring the number of microorganisms in a sample, discriminates microorganisms and foreign matters collected on a membrane filter, and accurately detects the number of microorganisms in the sample. An object of the present invention is to provide a microorganism detecting method and a microorganism measuring apparatus for detecting the number of microorganisms.

  In order to achieve the above object, the microorganism rapid detection method and the microorganism weighing apparatus of the present invention enable detection of microorganisms in a sample that may contain soymilk components and microorganisms by making the soymilk components finer than the size of the microorganisms. This is a microorganism detection method characterized by the above.

  Further, the present invention is a microorganism detection method for detecting microorganisms by filtration and extraction.

  In addition, the present invention provides a microorganism detection method in which soy milk components are finely divided to 0.2 μm or less.

  Further, the present invention provides a microorganism detection method for preventing aggregation after soymilk components are made fine.

  Further, the present invention is a method for detecting a microorganism in which a chelating agent is added to prevent aggregation.

  The chelating agent is a method for detecting microorganisms that is an aqueous solution of hexametaphosphoric acid.

  Moreover, it is set as the microorganisms detection method which adds 0.01-10% of the density | concentration of hexametaphosphoric acid with respect to the test substance which can contain a soymilk component and microorganisms.

  Further, the present invention is a method for detecting microorganisms to which an amide-based nonionic surfactant is added.

  Further, the present invention provides a microorganism detection method in which an organic solvent that enhances dispersibility is used as a solvent for an amide-based nonionic surfactant.

  In addition, the present invention provides a microorganism detection method characterized by providing a temperature that reduces the viscosity of a specimen that may contain soy milk components and microorganisms.

  Moreover, it is set as the microorganisms detection method characterized by adding the proteolytic enzyme.

  In addition, the present invention is a microorganism detection method in which an amide-based nonionic surfactant and a proteolytic enzyme are mixed and added.

  Further, the present invention provides a microorganism detection method characterized by using a heating means for promoting degradation after the addition of an amide-based nonionic surfactant and a proteolytic enzyme.

  Moreover, it is set as the microorganisms detection method characterized by making the temperature of the said heating means into 50-60 degreeC.

  In addition, the present invention provides a method for detecting microorganisms in which one or more ionic surfactants for reducing the viscosity are added to an amide nonionic surfactant and a proteolytic enzyme.

  In addition, the present invention provides a microorganism detection method characterized by using a fatty acid ester solvent with reduced moisture as a solvent for an amide-based nonionic surfactant and an ionic surfactant.

  In addition, the microorganism detection method includes a microorganism collection filter for filtering and extracting microorganisms, and means for adjusting pH so that the properties of the microorganisms do not change on the filter surface.

  Further, the present invention is a microorganism detection method comprising a microorganism collection filter for filtering and extracting microorganisms, wherein the filter is a filter whose surface shape is not deformed.

  In addition, the microorganism detection method includes a microorganism collection filter for filtering and extracting microorganisms, and the microorganism collection filter is a filter that does not lose color.

  Further, the present invention is a method for detecting microorganisms in which a pigment is supported on a microorganism collection filter.

  Further, the present invention is a microorganism detection method including a microorganism collection filter for filtering and extracting microorganisms, and the microorganism collection filter is a dark filter.

  Further, the present invention provides a microorganism detection method in which a thin film containing at least one metal component selected from gold, copper, chromium, platinum, and palladium is formed on a microorganism collection filter.

  In addition, a microorganism detection method in which microorganisms trapped on the microorganism collection filter are detected or counted by a staining method.

  The staining method is a microorganism detection method which is a fluorescence staining method.

  A first reagent in which the staining method reacts only with living cells to develop only living cells; a second reagent that reacts with only dead cells and develops only dead cells with a wavelength different from that of the coloring; A third reagent that reacts with any of the living and dead cells to develop a color at a wavelength different from that of the color development, and a color derived at a wavelength different from the color development by reacting with a microorganism-derived substance unique to the microorganism. The microorganism detection method uses any one kind or plural kinds of reagents among at least one kind of fourth reagents.

  In addition, after staining the microorganism with any one or a plurality of reagents among the first reagent, the second reagent, the third reagent, and the fourth reagent, a predetermined number is set. A light source for irradiating excitation light in a wavelength region, light receiving means for receiving a predetermined area of light set by the microorganism collection filter that emits light emitted by the excitation light, and light emitted by the light source. A microorganism judging means for receiving the emitted light within a predetermined time and judging that the received light quantity is within a set threshold value range and a set area range as a microorganism; In order to confirm the light emission point of one part or the entire area of the collection filter, the microorganism collection filter or the light receiving means is moved, the light emission point determined to be a microorganism by the microorganism determination means, 1 it is determined that, by sequentially accumulating, in which the microorganism detection method having means for accumulating the number of microorganisms.

  In addition, the light receiving means is a microorganism measuring device which is a plurality of photoelectric conversion elements having at least an area where the size of microorganisms can be recognized.

  The light source may be one type or a plurality of types, a predetermined wavelength range may be one type or a plurality of types, and light to be emitted emitted in the one type or a plurality of types of wavelength ranges may be determined in advance. The present invention provides a microorganism weighing device having a light receiving means for receiving light of a plurality of types of wavelength ranges, and a microorganism determination for determining a point or area of light emission from a ratio of light amounts in each wavelength range as a microorganism.

  In addition, the light source and the light receiving means of one kind or a set kind of light receiving means for receiving the plurality of types of light sources and the plurality of wavelength ranges are switched, and microorganisms are detected by the light source and the light receiving means of only the set types. It is a microorganism weighing device.

  Further, the microorganism judging means sequentially accumulates the areas of the light emitting points recognized as other than the microorganisms because of the set area or more, or recognizes the area as one and sequentially accumulates the number, and the total area or the total number is This is a microorganism weighing device having a caution means for indicating a caution when the set area or number is exceeded.

  Further, from the size of one light emitting point determined to be a microorganism by the microorganism determination means, it is determined that the mass of the microorganism, and the number of microorganisms in the size of the mass is converted and sequentially integrated to obtain the number of microorganisms. This is a microorganism weighing device having means for integrating.

  Further, the present invention is a microorganism measuring apparatus provided with means for determining the number of microorganisms and the number of microorganisms and displaying the number of microorganisms converted by the microorganism determining means.

  According to the present invention, in a sample containing a soy milk component, the soy milk component can be made finer than the size of the microorganism, and filter filtration is possible, whereby microorganisms are collected on the microorganism collection filter. It is possible to detect and measure microorganisms in a short time.

  In addition, it is possible to discriminate between microorganisms collected on the membrane filter and foreign substances, and to detect and measure the exact number of microorganisms in the sample.

  In addition, since the filter does not deform or decolorize the filter shape even when a surfactant is added to the specimen, it can be accurately measured without affecting the detection and measurement of microorganisms, even when measured by staining or fluorescent staining. Can detect and measure microorganisms.

  According to a first aspect of the present invention, there is provided a microorganism detecting method and a microorganism measuring device, wherein a sample containing soy milk components is made finer so that the soy milk components are smaller than the size of the microorganisms. The microorganism detection method is characterized by collecting microorganisms contained in the soy milk component on a filter and detecting the microorganisms, and has an effect of detecting microorganisms efficiently and with a simple configuration. Making soy milk components smaller than the size of microorganisms means that soy milk components are separated and dissolved, and soy milk components are made smaller than microorganisms. Dissolve the surroundings to reduce the size of the microparticles, disperse the aggregated microparticles to reduce the mass of the microparticles, prevent the soymilk component from becoming larger than the size of the microorganism due to reaggregation, etc. May be reduced, or liquefaction may be used.

  Further, the present invention is a method for detecting microorganisms by filtering and extracting microorganisms, and has an effect that the number of microorganisms in a sample amount can be measured by filtering.

  Also, it is a microorganism detection method in which microorganisms are filtered and extracted by making soymilk components into fine particles of 0.2 μm or less, and it is possible to remove impurities and accurately measure the number of microorganisms in a sample amount. Have

  Further, it is a microorganism detection method for preventing aggregation after soymilk components are microparticulated, and has an effect that measurement can be performed even if a micronized measurement specimen is left for a long time.

  In addition, this method is a microorganism detection method in which a chelating agent is added to prevent agglutination, and has the effect that measurement can be performed even if left for a long time without changing the properties of the micronized measurement sample.

  In addition, the chelating agent is a microorganism detection method characterized by adding an aqueous solution of hexametaphosphoric acid, and does not change the properties of the micronized measurement sample, and further identifies and chelates calcium in the sample. In addition, it suppresses changes in the shape of microorganisms contained in the specimen, and has an effect of measuring even if left for a long time.

  Moreover, it is a microorganism detection method in which the concentration of hexametaphosphoric acid is added to 0.01 to 10% with respect to a specimen that may contain soy milk components and microorganisms, and raw milk is obtained by specifying and chelating calcium in raw milk. It also suppresses changes in the shape of microorganisms contained in the specimen and has an effect of measuring even if left for a long time.

  In addition, it is an amide-based nonionic surfactant added, and has the effect of micronizing components other than microorganisms in soymilk.

  In addition, it is a microorganism detection method using an organic solvent that enhances dispersibility as a solvent for amide-based nonionic surfactants, and it improves the reactivity with the specimen and shortens the time required for micronization. Have

  In addition, it is a microorganism detection method characterized by providing a temperature that reduces the viscosity of a specimen that may contain soy milk components and microorganisms, and has the effect of further increasing the reactivity with the specimen and processing it in a short time. .

  Further, it is a method for detecting microorganisms by adding a proteolytic enzyme, and has an effect that proteins other than microorganisms can be decomposed and only microorganisms can be selected.

  Further, it is a microorganism detection method in which an amide-based nonionic surfactant and a proteolytic enzyme are mixed and added, and has the effect of reducing the number of samples and the number of mixing and improving workability.

  In addition, it is a microorganism detection method characterized by using a heating means that promotes degradation after the addition of an amide-based nonionic surfactant and a proteolytic enzyme. Of course, it has the effect of accelerating the degradation of proteins other than microorganisms and setting the measurement state in a short time.

  In addition, the microorganism detection method is characterized in that the temperature of the heating means is 50 to 60 ° C., enhances the enzyme reaction, promotes protein degradation and dissolution, reduces the viscosity of the measurement specimen, It has the effect of facilitating the handling of the specimen including handling.

  Further, the present invention is a microorganism detection method to which a surfactant and one or more types of ionic surfactants that lower the viscosity are added, and has an effect of lowering the viscosity of the specimen.

  In addition, the present invention is a microorganism detection method characterized by using a fatty acid ester solvent with reduced moisture as a solvent for amide-based nonionic surfactants and ionic surfactants. It has the effect of reducing the reaction and adapting to various specimens.

  In addition, the microorganism detection method includes a microorganism collection filter for filtering and extracting microorganisms, and a means for adjusting pH so that the properties of the microorganisms do not change on the filter surface. It has the effect of enhancing the sensitivity and detecting microorganisms with high sensitivity.

  In addition, the microorganism detection method includes a microorganism collection filter for filtering and extracting microorganisms, and the filter is a filter whose surface shape is not deformed, and improves the flatness of the filter surface to clarify the detection position of microorganisms. The device configuration at the time of detection / measurement can be simplified.

  The microorganism collection method is a microorganism detection method in which the filter for collecting microorganisms is a dark filter that does not lose color, and has the effect of suppressing the luminance of the background during fluorescence detection and increasing the sensitivity.

Further, it is a microorganism detection method in which a thin film containing at least one metal component selected from gold, copper, chromium, platinum, and palladium is formed on a filter for collecting microorganisms. In this case, it is possible to prevent reflection of light at the time, and to accurately detect and measure microorganisms without interfering with the detection and measurement of microorganisms.

  Further, it is a microorganism detection method for detecting or counting microorganisms captured on a microorganism collection filter by a staining method or a fluorescence staining method, and has an action of optically measuring emitted microorganisms.

  A first reagent that reacts only with living cells to develop color only of living cells; a second reagent that reacts only with dead cells and develops color of only dead cells with a wavelength different from the color development; A third reagent that reacts with any of the cells and causes any of the living and dead cells to develop color at a wavelength different from that of the color development; and at least develops a color at a wavelength different from the color development by reacting with a microorganism-derived substance specific to the microorganism One or more kinds of fourth reagents are brought into contact with the specimen, and the function of detecting both living cells and dead cells from the wavelength difference and the color development amount is provided. Have.

  In addition, after staining the microorganism with any one kind or plural kinds of reagents among the first reagent, the second reagent, the third reagent, and the fourth reagent, a predetermined wavelength is used. A light source that emits excitation light in a region; light receiving means that receives light of a predetermined wavelength region that is emitted by the excitation light and emits light; Microorganism judging means for receiving emitted light within a set time and determining that the received light quantity is within a set threshold range and within a set area range as a microorganism, and collecting the microorganism In order to confirm the light emission point of a part of the filter or the entire area, the microorganism collection filter or the moving means for moving the light receiving means, one light emitting point determined as a microorganism by the microorganism determining means, and the microorganism 1 It is determined that, by sequentially accumulating, which comprises means for integrating the quantity of microorganisms has the effect that differentiate weighed luminescent substance and microorganisms.

  Further, the light source is of one type or a plurality of types, the predetermined wavelength range is one type or a plurality of types, and the light to be emitted emitted in the one type or a plurality of types of wavelength ranges is determined in advance. The light receiving means for receiving light of each type of wavelength range and the function of detecting and detecting the point or area emitted from the ratio of the amount of light in each wavelength range as a microorganism.

  In addition, one type of light receiving means for receiving a plurality of types of light sources and a plurality of wavelength ranges or a set type of the light source and the light receiving means are switched, and microorganisms are detected by the light sources and the light receiving means for only the set types. Has an effect.

  In addition, by the microorganism judging means, the area of the light emitting points recognized as other than the microorganism because it is larger than the set area is sequentially accumulated, or the area is recognized as one and the number is sequentially accumulated, and the total area or the total number is There is a means for detecting when the area or number exceeds the set value and indicating attention.

  In addition, there is a means for accumulating the number of microorganisms by determining the mass of microorganisms from the size of one light emitting point determined to be a microorganism, converting the number of microorganisms in the size of the mass, and sequentially accumulating them. And has the effect of accurately measuring the microorganism concentration in the specimen.

  In addition, it has means for judging microorganisms and the mass of the microorganisms by the microorganism judging means and displaying them in each item, and has an effect that the microorganism concentration in the soymilk can be accurately measured.

  Hereinafter, embodiments of the present invention will be described with reference to the drawings.

(Embodiment 1)
For example, there are soy milk and various foods containing it as a raw material, but when the components contained in the soy milk perform filtration through a filter, the filter pores are blocked and filtration is inhibited. In addition, proteins themselves become fluorescent substances and are difficult to distinguish from microorganisms when observed with a microscope, and also interfere with observation of microorganisms. Usually, in order to measure microorganisms by the membrane filter method, filtration is performed with a 0.2 to 0.4 μm filter, and the microorganisms collected on the filter are observed with a microscope, or the filter is placed on a medium. Culture. In the food field, a filter having a pore size of 0.4 μm that can collect target microorganisms and has little influence on filtration is often used. That is, in order to filter soy milk, it is necessary to subdivide the components contained in the soy milk and filter and extract fine particles of 0.4 μm or less or completely dissolved (ionic) microorganisms.

  When filtration is performed with a 0.4 μm filter, the component of soy milk must be 0.4 μm or less, at least 0.2 μm or less, and preferably 0.1 μm or less. Of course, in reality, the particles are not uniformly 0.2 μm, but become fine with a certain normal distribution. Therefore, it is desirable that at least the center value of the particles is about 0.2 μm. If the maximum particle size is 0.2 μm or less, it can be applied to almost all filters, so it is more desirable if the center value is about 0.1 μm.

  The types of filters are PP (Polypropylene), PVC (Polyvinyl Chloride), PC (Polycarbon), PTFE (Polytetrafluorethylene), PVDF (Polyvinylidene fluoride), MCE (MelS). Depending on these filters, filtration is not always possible unless the thickness is 0.4 μm or less. For example, since MCE has flexibility in pores, some surface activity is required to improve filtration performance. Filtration is possible by filtering the soy milk after adding only the agent to the filter in advance, followed by heating (55 ° C) It made. However, in this method, the surface of the MCE is uneven, or microorganisms are buried in the pores, so that accuracy is insufficient when observing with a microscope.

  For example, since the surface is flat (± 2 μm or less, preferably ± 1 μm or less) as in the case of a PC material, and holes are made using radiation, and very uniform pores are formed, Since it is collected without being buried in the surface on the filter, it is usually used. Such a filter is poor in flexibility of pores and has a low aperture ratio, so that if there are particles having a particle size larger than the pores, it is difficult to filter. However, if substances and components other than microorganisms are made into particles with a particle size smaller than the pores, filtration becomes possible, and there are few irregularities on the surface and the microorganisms are not buried in the pores. It is also desirable for mechanical measurements.

  In addition, the components of soy milk include components that easily react with staining reagents and fluorescent staining reagents, and are very difficult and require skill when observed with a microscope or the like. By making these components also 0.2 μm or less, it becomes possible to distinguish them from microorganisms. Since general Escherichia coli, Staphylococcus aureus, etc. are about 0.5 μm in size, by making the components of soy milk 0.2 μm or less, it becomes possible to distinguish by size even if those particles are stained. . If the thickness is 0.1 μm, it becomes possible to distinguish more easily. However, even if it is 0.1 μm or less, if those components are covered on the filter, the entire filter will be stained, and it may be difficult to find the stained microorganisms, so that more accurate measurement is possible. It is desirable to decompose these components.

  Further, it was confirmed by microscopic observation that Escherichia coli and Staphylococcus aureus were collected by using a 0.4 μm filter. In addition, in the case where small bacteria such as Serthia are present, collection is possible by using a filter having a pore size of 0.2 μm.

  Therefore, various surfactants are mixed with raw milk to make soy milk components fine, and the PC filter pore size of 0.4 μm is used to check the filtration performance and micronization and interfere with measurement of various surfactants. It was confirmed whether the component remained. The confirmed result is shown in Table 1.

  In Tween 20, Tween 80, and Triton X-100, the liquid after the reaction process may or may not become a colored transparent liquid, so stable processing cannot be performed and sufficient reaction cannot be obtained. It is presumed that filtration becomes difficult and the treatment liquid becomes cloudy and large particles are present. In MEGA8, lipolane, RDA, and SDS, it was confirmed that the liquid after the reaction treatment became cloudy and filtration was difficult, and the residual effects of the components were large, greatly affecting the measurement. It is confirmed that amide-based nonionic surfactants are stable and can be filtered, the liquid after the reaction treatment is also stably processed into a transparent liquid, and that there are no substances that hinder measurement, soymilk It has been found that it is effective as a surfactant for making the component fine particles. Usually, soymilk is cloudy, but when mixed with an amide-based nonionic surfactant, it became transparent visually. Generally, when protein particles are mixed, the smaller the particles, the more transparent. Although it varies depending on the amount of particles, it is determined that the particle size is at least 0.2 μm or less, and since it is visually transparent, it seems that the particle size is about 0.1 μm. Ingredients contained in soy milk are often emulsions. In the present invention, a surfactant is used as a method of breaking or reducing protein particles, but physical means such as homogenization and ultrasonic waves, pH Even various chemical methods for causing changes or the like may be used as long as they do not dissolve or lyse microorganisms. This is because when lysed, the nucleic acid is eluted from the cell membrane or cell wall and cannot be detected. Of course, if the method that does not damage or affect the microorganism is the best method, the staining or fluorescent staining method can distinguish the life and death of the microorganism, so that the shape of the microorganism can be maintained and the surface of the microorganism can be dissolved. Any method that does not lyse can be measured.

  In addition, it was confirmed that the amide-based nonionic surfactant had no components that interfered with the measurement, and the microbial surface was dissolved or not lysed. This is because the particle diameter is 0.2 μm or less, and it is easy to distinguish microorganisms from other particles even when visually confirmed under a microscope.

  In addition, if left after reacting with an amide ion surfactant, the fatty acid in the soy milk component may recombine with calcium, reaggregate with calcium as the core, and change translucently. Transparency can be maintained even after the reaction by reacting the active agent with raw milk or adding a chelating agent that chelates calcium before and after the reaction. As chelating agents, ethylenediaminetetraacetic acid (EDTA), nitrilotriacetic acid (NTA) diethylenetriaminepentaacetic acid (DTPA), dicarboxymethyl glutamic acid tetrasodium salt (GLDA), hydroxyethylethylenediaminetriacetic acid (HEDTA), aminoethylethylene glycol ( GEDTA), triethylenetetramine hexaacetic acid (TTHA hexametaphosphoric acid), etc., and a concentration that does not affect microorganisms in soy milk is preferably about 0.01 to 10% of the amount of raw milk.

  However, even if the soymilk and the chelating agent are reacted and then reacted with the amide ion surfactant, if there is a lot of protein in the soymilk, the protein remains and may cover the microorganism collection filter for measurement. When fluorescent staining is performed, the fluorescent reagent remains in the protein and emits light although it is thin. When the microorganism collecting filter is covered, a means for degrading protein with an enzyme or the like may be used. It is possible to measure with higher accuracy by disassembling.

  Examples of proteolytic enzymes include proteinases, peptidases, trypsinogens, chymotrypsinogens, and the like, but they are capable of degrading proteins by giving a temperature although they have different active sites.

  In addition, when Triton X-100 or other surfactants are used in soy milk, many luminescent substances that appear to be other than microorganisms can be seen when judging the shape with a microscope, making it difficult to distinguish microorganisms and accurately measuring and measuring microorganisms. It can be difficult to do.

  In addition, depending on the type of amide-based nonionic surfactant, the viscosity may be high, and filtration may take time even if soy milk components are finely divided. In water, the viscosity does not decrease, but it may become cloudy and make filtration difficult. In such a case, the addition of an ionic surfactant further reduces the viscosity and improves the filtration performance. There are five types of ionic surfactants: 1) fatty acid (anion), fatty acid sodium, fatty acid potassium, alpha sulfo fatty acid ester sodium, 2) linear alkyl benzene, linear alkyl benzene sulfonic acid Sodium, 3) Higher alcohols (anions), sodium alkyl sulfates, sodium alkyl ethers, 4) Alpha olefins, sodium alpha olefin sulfonates, 5) Normal paraffins, sodium alkyl sulfonates, etc. However, any reagent may be used as long as it does not cause a chemical reaction with the amide-based nonionic surfactant and does not dissolve or lyse microorganisms.

  Also, depending on the amount of water in the soymilk, mixing an aqueous solution of an amide non-surfactant and an ionic surfactant may promote reagglomeration and become cloudy. The amide non-surfactant and ionic surfactant It is necessary to reduce the amount of the aqueous solution. By using fatty acid ester glycerin, propylene glycol or the like instead of the aqueous solution, the influence of moisture can be reduced, re-aggregation can be prevented, and white turbidity can be prevented.

  After filtering, when dyed or fluorescently stained microorganisms are observed with a microscope and measured with an automated apparatus, it is possible to measure with high accuracy by using a dark filter that does not lose color. A dark color is black or a color close to it, but is a color that does not absorb light emitted when irradiating light of a specific wavelength according to the characteristics of the reagent, such as white light or ultraviolet light, when observing with a microscope. It is shown that. Many of the materials of the filter shown above are white or close to white, and particularly when irradiated with ultraviolet rays, reflection or autofluorescence may cause the measurement to be difficult. When the filter gets wet with water, reflection hardly occurs, but it is important as a color of the filter that the reflection is prevented when wet with water. Usually, since the material of the filter is rarely dark, it needs to be colored in black or gray that is close to black, and as its method, a pigment or ink that does not easily lose color is desirable.

  When a surfactant is used when making protein particles into fine particles, the color tends to be lost due to the influence of the surfactant, and as a result, the material portion of the filter is exposed and cannot be measured. Reflection can be prevented by coating the filter surface with a metal thin film selected from gold, copper, chromium, platinum, palladium and the like by vapor deposition or the like. Gold, in particular, has a low ultraviolet reflectance. In other words, the dark color indicates a color close to black and has little light reflection. Further, even when a solvent that dissolves like a surfactant is used, the metal is not dissolved and the filter material is not exposed, so that measurement is not disturbed.

  As a method for staining microorganisms, 4 ′, 6-diamidino-2-phenylindole dihydrochloride as a first reagent and propium iodide as a second reagent and a second reagent on a sample to which cells or microorganisms are attached are used. The reagent which mixed 6-carboxyfluorescein diacetate which is the reagent 3 and 4-methylumbelliferyl-β-D-galactoside which is the fourth reagent is brought into contact. The 4 ′, 6-diamidino-2-phenylindole dihydrochloride bound to the nucleic acid of the living and dead cells absorbs the excitation wavelength when the excitation wavelength is 359 nm and emits a fluorescence wavelength of 461 nm to cause the living and dead cells to develop color. Propidium iodide combined with dead cell nucleic acid absorbs the amount of light at the excitation wavelength when the excitation wavelength is 535 nm and changes the fluorescence wavelength to 617 nm to cause only dead cells to develop color.

  Other fluorescent reagents for live and dead cells include YTO11 to SYTO16, SYTO20 to SYTO25, SYTO40 to SYTO45, SYTO17, SYTO59 to SYTO64, SYTO80 to SYTO85, DAPI, SYBRGreen, Hoechst33342, HoechY33TO, 58 . Examples of fluorescent luminescence reagents for dead cells include Propium Iodide, SYTOXGreen, BOBO-1, YOYO-1, YO-PRO-1, TOTO-1, POPO-3, TO-PRO3, SYTOXBlue, and SYTOXOrange. By setting the wavelength of the excitation light with the fluorescent reagent, the living and dead cells and dead cells that have reacted with the reagent can emit fluorescence.

  In addition, those that react with esterase that only live bacteria have, those that stain only live bacteria using a reagent that can detect respiratory activity, and those that die because the cell membrane is damaged because it does not penetrate the cell membrane, Permeated from there and stained with nucleic acid such as DNA to stain only dead bacteria. Similarly, it binds to nucleic acid such as DNA and has the property of permeating the cell membrane. Those that stain both bacteria, those that react with specific microorganism-derived substances that only specific microorganisms metabolize, antibodies that have fluorescent labels that react only with specific microorganisms, or those that stain only specific microorganisms with microphages, etc. There is.

  When conducting microbiological tests on dairy products, the larger the sample amount, the better. In the conventional bleed method, since 0.01 ml is the maximum, the amount of the sample can be 0.1 ml or 1 ml or more by filtration, and the bacteria concentration is measured as the number per 1 ml. The greater the number, the better the sensitivity and accuracy.

(Embodiment 2)
FIG. 1 is a block diagram showing an embodiment of a microorganism weighing device. This microorganism weighing device includes a lens 1 and a light receiving unit 2 as light source condensing means. In order to extract the target wavelength from the excitation light emitted from the light source 3, the light is spectrally separated by the excitation light spectral filter 4. The split excitation light passes through the prism 5 and the optical path is changed. The excitation light whose optical path has been changed passes through the lens 1 and includes a microorganism collecting filter 7 installed on the examination table 6, that is, a combined microorganism collecting filter 7 composed of the microorganism collecting filter 7 and the pedestal 8. Focused on the surface. Therefore, the fluorescence of the microorganisms excited by the excitation light passes through the prism 5 again and reaches the light receiving unit 2. The fluorescence that has reached the light receiving unit 2 passes through the fluorescence spectroscopic filter 9 in order to extract only the target fluorescence, reaches the photoelectric conversion element 10 built in the light receiving unit, is converted into a signal, and is recognized. Although not shown in the figure, this microorganism weighing device includes means for moving the inspection table 6 and can receive all or part of the fluorescence emitted from the surface of the filter 7 for collecting microorganisms.

  In the fluorescence of 461 nm and 617 nm that reached the photoelectric conversion element 10, viable bacteria and dead bacteria emitted fluorescence of 461 nm, and dead bacteria emitted fluorescence even at 617 nm. The difference in the wavelength of light and the light wavelength is separated by the fluorescence spectral filter 9 and only the target fluorescence wavelength is extracted and reaches the photoelectric conversion element 10, which absorbs the excitation wavelength 359nm and is excited to emit fluorescence at 461nm. And those that are excited and absorb fluorescence at 617 nm and emit fluorescence at 617 nm are judged as dead bacteria, and those that do not emit fluorescence at both 461 nm and 617 nm are judged as foreign substances. The fluorescence determined to be microorganisms is integrated and the quantity is measured. The microorganism judging means 11 includes a microcomputer programmed with the judging procedure.

  The excitation light generated from the light source 3 is condensed by the lens 1, and at this time, the range irradiated with the excitation light by the lens 1 is condensed on a small fixed area. In this case, the small fixed area indicates a range of about 0.2 μm to 7.0 μm on a side when set based on the size of the microorganism. In addition, when based on a comparison with the agar medium diffusion method, which is one of the most utilized microorganism detection means, colonies formed by a population of microorganisms cultured and grown by the agar medium diffusion method have a distance of When they are close to each other, colonies may overlap with each other, and when they are finally visually confirmed, there may be cases where the colonies are recognized as one colony. Therefore, the minute fixed area in this case indicates a range of about 100 μm to 500 μm on a side when based on a distance where colonies do not overlap each other. That is, the small fixed area is a size that can be recognized as a microorganism by the microorganism determination means 11. The light receiving unit 2 may shoot the entire filter as a single image with a plurality of photoelectric conversion elements 10, or after shooting with a small photoelectric conversion element 10 (one side is 10 μm or less), each image is assembled. In addition, the size of the microorganism may be finally recognized.

  The irradiation time of the excitation light collected by the lens 1 depends on the quenching time and the excitation light intensity of the reagent that emits fluorescence. Depending on the type of reagent, it may be decomposed by ultraviolet light existing in nature, and it is desirable to irradiate excitation light within a range of about 2 to 300 seconds.

  When light emission is detected, if the wavelength range of the light source 3 is wide, the excitation wavelength can be adjusted and dispersed by the excitation light spectral filter 4. Since the excitation light spectral filter 4 can be changed according to the target detection target, it can cope with various fluorescent reagents.

  At the same time, when the emitted fluorescence wavelength has a wide range, it is possible to cope with various fluorescent reagents by changing the fluorescence spectral filter 9 according to the target detection target in order to detect the target emission. .

  Examples of the light source 3 include various diodes, halogen lamps, xenon lamps, cold cathode tubes, lasers, black lights, mercury lamps, and the like. Among these light sources, a diode, a cold cathode tube, a black light, or the like whose maximum excitation wavelength is relatively limited may be implemented without using the excitation light spectral filter 4 and the fluorescence spectral filter 9. For halogen lamps, mercury lamps, and the like, it may be necessary to use the excitation light spectral filter 4 and the fluorescent spectral filter 9.

  The prism 5 and the lens 1 each have a property of transmitting ultraviolet light as necessary. Quartz glass etc. are mentioned as what has the property which permeate | transmits ultraviolet light. Thereby, it can respond also to the reagent etc. which are excited by ultraviolet light. The inspection table 6 on which the part including the filter 7 for collecting microorganisms has a rotating ability. The excitation light collected by the lens 1 moves a distance corresponding to the radius from the outer periphery of the microorganism collection filter 7 to the center or from the center to the outer periphery. At that time, the excitation light condensed by the lens 1 is changed by changing the rotation speed of the examination table 6 when the position of the excitation light collected by the lens 1 is present at the outer peripheral portion and at the central portion. It is possible to prevent the occurrence of deviation in fluorescence, afterimage and afterglow generated by the excited reagent when the is present at the outer periphery and at the center.

  As shown in FIG. 1, the inspection table 6 has a depressed portion (apparatus groove) for fitting a part including the microorganism collecting filter 7, and the part including the microorganism collecting filter 7 can be directly incorporated therein. It can be shaped. At this time, for example, the inspection table 6 is provided with a metal flat plate so that the microorganism collecting filter 7 is positioned thereon, and the microorganism collecting filter 7 is incorporated in a state of being pressed against the metal flat plate. Thus, if the microorganism collection filter 7 is held smoothly on the inspection table 6 without unevenness, the quantification of the microorganisms collected in the microorganism collection filter 7 can be made more reliable.

  It should be noted that by providing means for recognizing the condensed position, the position of the excitation light collected by the lens 1 is recognized, so that the condensed light does not deviate from the orbit, and when it deviates, it returns to the orbit again. Is set to

  Note that the minute fixed area to which the excitation light is irradiated is not limited to a polygon including a square, but may be a circle, an ellipse, or the like as long as the sample can be irradiated.

  It is also possible to use a diffraction grating or the like as means for separating excitation light or fluorescence.

  Although the fluorescence afterimage and afterglow are prevented by adjusting the rotation speed of the inspection table 6, it is also possible to prevent afterimage and afterglow by adjusting the moving speed of the lens 1 that irradiates the excitation light. It is.

  The means for recognizing the condensed position is not necessarily required to directly recognize the condensing position of the excitation light, and may be any means that can grasp the trajectory on the microorganism collection filter 7.

  FIG. 2 is a flowchart for making the soy milk component finer than the size of the microorganism in the sample that may contain the soy milk component and the microorganism of the present invention. Soymilk was used as a specimen that may contain soymilk components and microorganisms.

  As a method for micronizing soy milk components, 1 ml of soy milk is collected in step 1 and drawn into a reaction vessel. Further, 0.1 ml of reagent A is added. In step 2, the whole liquid is mixed with a stirrer so that the soy milk and reagent A are sufficiently brought into contact with each other in the liquid. As reagent A, hexametaphosphoric acid was used.

  As Step 3, 0.1 ml of the mixed solution of Step 2 is collected and 0.8 ml of Reagent B is added. Reagent B used was amide nonionic surfactant amizole. About 3% of the amisole reagent was mixed with the soymilk. In Step 4, the whole liquid is mixed with a stirrer so that the raw milk and amizole are sufficiently brought into contact with each other in the liquid. By step 5, the liquid in which the components in the soymilk are finely divided and clouded becomes a nearly transparent liquid. In step 5, 1 ml of reagent C for further microparticulation is added to the treatment liquid. Reagent C used was a protease (Bacillus licheniformis Protease). For the protease concentration, a 10% aqueous solution is used. As Step 6, the whole liquid is mixed with a stirrer so that the treatment liquid (Step 4 treatment liquid) and protease are sufficiently contacted. In step 7, incubation is performed at a constant temperature and time to activate the reaction by the protease. Although the temperature at this time shall be 50 to 60 degreeC, around 55 degreeC is preferable. The processing time is 3 to 10 minutes, but about 3 minutes is preferable. In step 8, the processing liquid is mixed with a stirrer to complete the micronization processing. Further, the liquid that has become cloudy due to the above configuration becomes a yellow transparent liquid, and the soy milk component is finely divided.

  In the specimen staining, the whole amount of 1.9 ml of the treatment liquid is filtered through the microorganism collection filter 7 in Step 9. In step 10, residual components other than microorganisms on the surface of the microorganism collection filter 7 are washed away with 3 ml of physiological saline. In step 11, 0.1 ml of reagent D, which is a fluorescent staining reagent for staining the microorganisms collected by the microorganism collection filter 7, is dropped, and the reagent D is spread over the entire surface of the microorganism collection filter 7 and stained for 2 minutes. . Reagent D was a reagent that binds to and stains microbial DNA. In step 12, the reagent D surplus reagent is washed with 0.1 ml of physiological saline to complete the specimen staining.

  In the measurement, the microorganism collection filter 7 is set on the base of the measuring device in step 13. About this stand, it is comprised in the state which becomes a plane by setting the filter 7 for microorganisms collection on a measurement surface, and can thereby perform the stable measurement. In step 14, the microorganisms in the collected soymilk are measured by the microorganism measuring device, and the measurement is completed.

  FIG. 5 shows an image of fluorescent emission after fluorescent staining after completion of the pretreatment. As shown in the figure, the fluorescence emission point has various sizes, and the minute size was observed with a microscope, and as a result, it was confirmed that the number of cells was one. It was also confirmed that the fluorescence emission points having a size other than the minute light-emitting substance were streptococcus with solid bacterial cells. It became clear that what was captured with the filter for collecting microorganisms after completion of the pretreatment can indicate contamination in soy milk.

  FIG. 3 shows a flow chart for measuring microorganisms in an image of one field taken by the microorganism testing apparatus of FIG. When measuring a large number of fields of view, it is possible to calculate the number of microorganisms in the entire filter by repeating this flow and multiplying the ratio of the area of the entire filter and the measured area (field of view area × number of fields of view) by the number of microorganisms measured. it can.

  The results of this example are shown in FIG. It was found that there is a correlation between the conventional method and the method of the present invention.

  In addition, although the density | concentration which mixed the amizole and soymilk was 3% in the Example, even if it uses in the range of 0.03% to 6%, the same effect is acquired.

  In addition, although it does not describe in the present Example about the heating method about incubation, if a reagent and soymilk can be heated with a thermostat, a heater, an ultrasonic wave, an electron beam, etc., there will be no problem in particular. Can be used.

  Although it has been described that the treatment time is preferably about 3 minutes, the property with the mixture is not particularly changed even if the treatment is performed for a long time.

  In addition, although it described as the liquid near transparency, it is a pale or transparent thing at the visual level, and shows what became the fine particle of 0.2 micrometer or less.

  In the examples, an aqueous solution having a protease concentration of 10% was used, but a concentration of 1% to 15% may be used.

  In the examples, an aqueous protease solution was used, but a solvent dissolved in a solution having a pH of 5 to 8 may be used.

  In the examples, an aqueous protease solution was used, but even if propylene glycol or glycerin is used as the organic solvent, there is no difference in performance.

  In the embodiment, the cleaning removal is performed in step 10, but the cleaning removal process can be omitted particularly when the measurement accuracy of microorganisms is set low.

  The reagent D used is a reagent that binds to and stains microbial DNA, but an antibody reaction, a metabolic enzyme reaction, or a respiratory reaction reagent may be used.

  In addition, although the single dyeing | staining was implemented as the reagent D in the Example, double dyeing | staining can also be carried out.

  In addition, although the washing | cleaning process of the excess reagent was described in the Example, the process of washing | cleaning an excess reagent can be excluded if there is no influence on the background at the time of a measurement.

  In the examples, soymilk was used as a specimen, but it can also be used for an aqueous solution containing soymilk components.

  In addition, although it stirred using the stirrer at the time of mixing, if a reagent and a test substance can be stirred, there will be no difference in particular.

  The microbe detection method and the microbe measuring apparatus for detecting microorganisms by making the soymilk component finer than the size of the microbe in the specimen that may contain the soymilk component and the microorganisms of the present invention do not require skilled skills, and anyone Since it can be measured easily, it can be applied to quality control at a location closer to the production site. In recent years, the quality level of food has been improved, and there is a need for a device that can measure the number of microorganisms quickly, with high accuracy, and easily at low bacterial count levels, and can be applied as a method to replace conventional tests. .

Configuration diagram showing one embodiment of microorganism detection method and microorganism light weight device Flow chart showing reaction configuration of microorganism detection method and microorganism light weight device Microorganism detection method and measurement flow chart of microbial lightweight device A graph showing the correlation between the microorganism detection method and the conventional method of the microorganism lightweight device Micrograph of fluorescence emission image of Example 1 of microorganism detection method and microorganism light weight device

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Lens 2 Light-receiving part 3 Light source 4 Excitation light spectral filter 5 Prism 6 Inspection stand 7 Microbe collection filter 8 Base 9 Fluorescence spectral filter 10 Photoelectric conversion element 11 Microorganism judgment means

Claims (32)

A microorganism detection method characterized in that microorganisms can be detected in a specimen that may contain soy milk components and microorganisms by making the soy milk components finer than the size of the microorganisms. The microorganism detection method according to claim 1, wherein the microorganism is detected by filtration extraction. The method for detecting microorganisms according to claim 1 or 2, wherein the soymilk component is finely divided into particles of 0.2 µm or less. The microorganism detection method according to any one of claims 1 to 3, wherein aggregation is prevented after the soymilk component is finely divided. The microorganism detection method according to claim 4, wherein a chelating agent is added to prevent aggregation. The method for detecting a microorganism according to claim 5, wherein the chelating agent is an aqueous solution of hexametaphosphate. The microorganism detection method according to claim 6, wherein the concentration of hexametaphosphoric acid is added in an amount of 0.01 to 10% with respect to a specimen that may contain soy milk components and microorganisms. The microorganism detection method according to any one of claims 1 to 7, wherein an amide-based nonionic surfactant is added. The microorganism detection method according to claim 8, wherein an organic solvent that enhances dispersibility is used as a solvent for the amide-based nonionic surfactant. The method for detecting a microorganism according to any one of claims 1 to 9, wherein a temperature is applied to reduce the viscosity of a specimen that may contain soy milk components and microorganisms. The microorganism detection method according to any one of claims 1 to 10, wherein a proteolytic enzyme is added. The microorganism detection method according to any one of claims 1 to 11, wherein an amide-based nonionic surfactant and a proteolytic enzyme are mixed and added. 13. The microorganism detection method according to claim 12, wherein a heating means for promoting the decomposition is used after the addition of the amide-based nonionic surfactant and the proteolytic enzyme. The microorganism detection method according to claim 13, wherein the temperature of the heating means is set to 50 to 60 ° C. The microbial detection method according to any one of claims 1 to 14, wherein one or more ionic surfactants for reducing the viscosity are added to the amide nonionic surfactant and the proteolytic enzyme. 16. The method for detecting microorganisms according to claim 15, wherein a solvent for fatty acid esters with reduced water content is used as the solvent for the amide-based nonionic surfactant and the ionic surfactant. The microorganism detection method according to any one of claims 1 to 16, further comprising a microorganism collection filter for filtering and extracting microorganisms, and means for adjusting pH so that the properties of the microorganisms do not change on the filter surface. The microorganism detection method according to claim 17, further comprising a filter for collecting microorganisms for filtering and extracting microorganisms, wherein the filter is a filter whose surface shape is not deformed. The microorganism detection method according to claim 17 or 18, further comprising a microorganism collection filter for filtering and extracting microorganisms, wherein the filter is a filter that does not lose color. 20. The microorganism detection method according to claim 18 or 19, wherein a pigment is supported on the microorganism collection filter. The microorganism detection method according to any one of claims 18 to 20, further comprising a microorganism collection filter for filtering and extracting microorganisms, wherein the filter is a dark filter. The microorganism detection method according to any one of claims 18 to 21, wherein a thin film containing at least one metal component selected from gold, copper, chromium, platinum, and palladium is formed on the microorganism collection filter. The microorganism detection method according to any one of claims 17 to 22, wherein microorganisms captured on the microorganism collection filter are detected or counted by a staining method. The microorganism detection method according to claim 23, wherein the staining method is a fluorescence staining method. A first reagent that causes only living cells to develop color by reacting only with living cells, a second reagent that reacts only with dead cells and develops only dead cells at a wavelength different from the color, A third reagent that reacts with any of the viable and dead cells to develop a color at a wavelength different from that of the color development, and at least one kind that develops a color at a wavelength different from the color development by reacting with a microorganism-derived substance unique to the microorganism The microorganism detection method according to claim 23 or 24, wherein any one kind or plural kinds of reagents are used among the fourth reagents. After staining the microorganism with one or more of the first reagent, the second reagent, the third reagent, and the fourth reagent, in a predetermined wavelength range A light source for irradiating excitation light; light receiving means for receiving light of a predetermined wavelength range emitted by the excitation light and receiving a predetermined area set by the microorganism collection filter; and light emitted by the light source. A microorganism judging means for receiving light within a set time and determining that the received light quantity is within a set threshold value range and a set area range as microorganisms; and In order to confirm the light emission point of one part or the entire area, it is determined that the microorganism collection filter or the light receiving means is moved, a light emission point determined as a microorganism by the microorganism determination means and one microorganism. To, by sequentially accumulating microorganisms weighing apparatus for performing the microorganism detection method of claim 25, further comprising means for integrating the quantity of microorganisms. 27. The microorganism measuring apparatus according to claim 26, wherein the light receiving means is a plurality of photoelectric conversion elements having an area where at least the size of microorganisms can be recognized. There are one type or a plurality of types of light sources, one type or a plurality of types of predetermined wavelength ranges, and a plurality of types of light that is emitted in one or more types of wavelength ranges. 28. The microorganism measuring apparatus according to claim 26 or 27, comprising: a light receiving means for receiving light in each wavelength region; and a microorganism determination for determining a light emission point or area as a microorganism from a ratio of light amounts in each wavelength region. 27. The microorganisms are detected by the light source and the light receiving means of only the set type by switching the light source and the light receiving means of one type or a set type of light receiving means for receiving a plurality of types of light sources and a plurality of wavelength ranges. The microorganism weighing device according to any one of items 28 to 28. The microorganisms judging means sequentially accumulates the areas of the light emitting points recognized as other than microorganisms because it is larger than the set area, or recognizes the area as one and sequentially accumulates the number, and the total area or the total number is the set area The microorganism weighing device according to any one of claims 26 to 29, further comprising a caution means for indicating a caution when the number is more than the number. Means for accumulating the quantity of microorganisms by judging the mass of microorganisms from the size of one light emitting point judged to be a microorganism by the microorganism judging means, converting the number of microorganisms in the size of the mass, and sequentially integrating the quantities. A microorganism weighing device for performing the microorganism detection method according to any one of claims 26 to 30. 33. The method for detecting a microorganism according to any one of claims 26 to 32, comprising means for determining a microorganism, a mass of microorganisms, and a somatic cell by a microorganism judging means, and displaying the number of microorganisms, the number of microorganisms converted, and the number of somatic cells. Microorganism weighing device.

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