JP5958998B2 - Spore measurement method - Google Patents

Description

  The present invention relates to a method for introducing substances into spores and a method for measuring spores having germination activity using the same.

  Spores formed by spore bacteria such as Bacillus and Clostridium and molds such as Aspergillus and Penicillium have high heat resistance, chemical resistance, and drought resistance, and are difficult to sterilize. Therefore, in the food manufacturing industry, the pharmaceutical manufacturing industry, etc., contamination by the above spores often becomes a problem.

  In the production of foods, pharmaceuticals, etc., the number of spores present in products, intermediate products, raw materials or production environment (hereinafter sometimes referred to as spore count) is monitored, and product sterilization and production environment cleaning are appropriately performed. It has been confirmed that raw materials are not excessively contaminated by spores and the like.

JP 2006-174751 A JP 2007-209223 A JP-A-6-165667

Kenji Nishi, Ryo Kato, Mamoru Tomita, "Activation and disinfection of Bacillus spore by pressure", Japan Society for Food Science and Technology 41 p.542-549 1994 Aoyama, Y., Shigeta, Y., Okazaki, T., Hagura, Y. and Suzuki, K, “Germination and Inactivation of Bacillus subtilis Spores under Combined Conditions of Hydrostatic Pressure and Medium Temperature”, Food Sci. Technol. Res. , 11 (1), p.101-105. 2005 J. G. CLOUSTON AND PAMELA A. WILLS, “Initiation of Germination and Inactivation of Bacillus pumilus Spores by Hydrostatic Pressure”, JOURNAL OF BACTERIOLOGY, Feb. 1969, p.684-690

  Usually, the number of spores is monitored by a culture method in which spores are cultured using a medium and the number of colonies formed by the cultured spores is confirmed. Since spore culture takes a long time, the culture method requires a period of 2 to 14 days until the results are known. Therefore, if abnormalities such as contamination of the product are detected as a result of monitoring, the response to improvement measures such as cleaning and sterilization will be delayed by the period for which the results are known. Development of a measurement method is desired.

  Patent Document 1 discloses that the number of spores is measured by the following method. First, a spore having a hard outer wall is germinated by culturing in a liquid medium for 2 hours or standing at 2 ° C. to 25 ° C. for 6 hours to 20 hours to change to a vegetative cell having no hard outer wall. Thereafter, the vegetative cells are stained with a fluorescent dye, and the spore count is measured by counting the stained vegetative cells. Furthermore, Patent Document 1 discloses a method of measuring the number of spores by subjecting spores to heat shock treatment at 60 ° C. to 100 ° C. for germination and then staining and counting vegetative cells with a fluorescent dye.

  However, since it is unclear what kind of genus microorganism is contained in the sample, the method of culturing spores in the liquid medium described in Patent Document 1 selectively selects vegetative cells of various genus species. It tends to be difficult to change. If the selection of antibacterial substances to be added to the liquid medium is not appropriate, spores may not germinate and change to vegetative cells. In addition, in this method, since it takes several hours to germinate spores, not only measurement takes time, but also the spores and microorganisms other than the spores contained in the sample grow, and the accurate spore count and microorganisms There is a tendency that the number cannot be measured.

  In addition, since heat shock treatment has a low germination-inducing effect, germination is not induced quickly only by heat shock treatment in many cases, and an accurate measurement of the number of spores by introducing a fluorescent dye inside the spores Tend to be difficult.

  On the other hand, Patent Documents 1 and 2 disclose that a germination substance such as alanine is added to a spore to cause germination and change to a vegetative cell. In Patent Document 1, vegetative cells are then stained with a fluorescent dye to measure the number of spores. In Patent Document 2, vegetative cells are then lysed for gene extraction and gene analysis.

  However, germination-inducing substances such as alanine have low germination-inducing effects, and the effects of each germination-inducing substance differ depending on the spore genus species. When measuring the number of spores, it tends to be difficult to select an appropriate germination inducer.

  It is also known that spore germination is induced by pressurization. Germinated spores lose their heat resistance and chemical resistance by losing their outer walls and can be easily sterilized, so a sterilization method using germination of spores by pressurization has been reported (Non-Patent Document) 1-3).

  However, since pressurization of spores is aimed at sterilizing spores, life and death of spores and other microorganisms due to pressurization are not considered, and attempts to introduce substances into the spores by germination Nor.

  The present invention has been made in view of the above circumstances, and can be applied to spores of a wide range of genus species in a short time, and a method for introducing substances into spores and the number of spores having germination activity using the method. The purpose is to provide a measurement method.

The present invention is a pressure treatment step of pressurizing the target spores at a pressure of 10 MPa to 400 MPa at a temperature of 5 ° C. to 70 ° C. for 1 second to 60 minutes,
A substance mixing step of mixing the spore and a substance to be introduced into the spore;
The substance mixing step provides a method for introducing a substance into a spore, which may be before or after the pressure treatment step.

  Under such pressure and pressure conditions, the material permeability of spores of a wide range of species can be improved in a short time, and the target substance can be introduced into the spores. That is, by appropriately setting the pressure and temperature, germination of spores of various genera can be efficiently induced in a short time, and the substance introduction effect into the inside of the spores is significantly improved.

  The substance is preferably a dye, protein, oligonucleotide, plasmid, compound containing a radioisotope, or ion.

  It is preferable that the said substance introduction | transduction method performs a pressurization process process in presence of the germination inducer with respect to a spore. By performing the pressure treatment in the presence of a germination inducer, the germination rate of spores is further improved, and the substance introduction effect is further improved.

  Preferably, the substance introduction method further includes an atmospheric pressure treatment step of holding the spores at a pressure of 5 ° C. to 70 ° C. for 1 to 60 minutes after the pressure treatment step. Thereby, the germination rate of a spore further increases and the substance introduction effect is further improved.

  Moreover, this invention provides the measuring method of the spore which has germination activity provided with the process of introduce | transducing the substance used as a marker into the target spore by the said substance introduction | transduction method.

  According to such a method, a marker substance can be introduced into spores of a wide range of genus species in a short time, and the number of spores having germination activity can be measured. In the present invention, since the spores having germination activity are only living spores, in the sterilized food, etc., not the number of dead spores but the number of living remaining spores that can grow in the distribution process is measured. it can.

  According to the present invention, it is possible to provide a method for introducing a substance into a spore that can be applied to spores of a wide range of genus species in a short time, and a method for measuring the number of germinating spores using the method. .

It is a schematic diagram of the pressure heating apparatus which concerns on this embodiment.

  Hereinafter, embodiments of the present invention will be described in detail. However, the present invention is not limited to the following embodiments.

  The substance introduction method to the spore according to the present embodiment includes a pressure treatment step in which the target spore is pressurized at 10 to 400 MPa at a temperature of 5 ° C. to 70 ° C. for 1 second to 60 minutes, and the spore And a substance mixing step of mixing the substance to be introduced into the spore. The substance mixing step may be before or after the pressure treatment step. By applying the pressure treatment as described above to the spore, it becomes possible to improve the permeability of a substance such as a fluorescent dye into the spore.

  The spore according to the present embodiment means a spore produced by a general spore fungus or mold existing in a food or natural environment. Examples of the spores include, for example, Bacillus such as Bacillus cereus and Bacillus subtilis, Paenibacillus polymyxa, Paenibacillus macerans and Paenibacillus macerans. Paenibacillus alvei, Paenibacillus, Geobacillus stearothermophulus, etc., and Clostridium botulinum, Clostridium pertrins, etc. Spores produced by spore bacteria such as Clostridium, and Aspergillus and Penicillium digitatum, such as Aspergillus niger Penicillium digitatum) is Penicillium (Penicillium) mold such as include spores produced. The spore may be used as it is, but is preferably used in a state suspended in an appropriate buffer solution or the like.

  In the pressure treatment process according to this embodiment, the temperature of the pressure treatment is 5 ° C to 70 ° C. The temperature is preferably 5 ° C. to 50 ° C., more preferably 30 ° C. to 50 ° C., from the viewpoint of reducing the cost of the apparatus and preventing the inactivation of spores by the pressure treatment. If the temperature is less than 5 ° C., the spore germination cannot be promoted sufficiently, and as a result, the substance introduction effect (substance permeability) tends to be insufficient. When the temperature exceeds 70 ° C., spores with relatively low resistance tend to die.

  In the pressure treatment process according to the present embodiment, the pressure of the pressure treatment is 10 MPa to 400 MPa, preferably 10 MPa to 200 MPa, and more preferably 50 MPa to 100 MPa. By setting the pressure in such a range and the temperature in the above range, spores of a wide range of genus species can be effectively germinated in a short time. As a result, the substance permeability of the outer wall of the spore is improved, and the target substance can be easily introduced into the spore. If the pressure is less than 10 MPa, spore germination cannot be promoted sufficiently, and as a result, the substance introduction effect (substance permeability) tends to be insufficient. When the pressure exceeds 400 MPa, spores with relatively low resistance tend to die. Examples of the pressurizing method include a method using a hydrostatic pressure and a pressure through a medium such as a gas pressure and a hydraulic pressure, and a method using a hydrostatic pressure is preferable. Examples of the pressurization method include a batch method, a semi-batch method, and a continuous method. You may pressurize by centrifugation.

  In the pressure treatment process according to the present embodiment, the pressure treatment time is 1 second to 60 minutes, preferably 1 minute to 60 minutes, and more preferably 1 minute to 15 minutes. When the time exceeds 60 minutes, some spores tend to die, and rapidity tends to decrease.

  The pressure treatment process according to the present embodiment is performed using, for example, an apparatus shown in FIG. FIG. 1 is a schematic diagram of a pressure heating apparatus according to the present embodiment. The pressure heating apparatus 10 includes a pressure vessel 1 for containing a sample to be pressurized, a heater 2 that controls the temperature inside the pressure vessel 1, a pressure pump 3 that controls the pressure inside the pressure vessel 1, A temperature sensor 4 for monitoring the temperature inside the pressure vessel 1 and a pressure gauge 5 for monitoring the pressure inside the pressure vessel 1 are provided. The pressure vessel 1 is, for example, a cylindrical closed vessel having an inner diameter of 100 mm, and the wall thickness dimension is set to 150 mm. For example, the heater 2 can raise the temperature inside the pressure vessel 1 to 90 ° C., and can set the inside of the pressure vessel 1 to an arbitrary temperature. The pressurization pump 3 can adjust the inside of the pressure vessel 1 to an arbitrary pressure up to 500 MPa, for example. Further, the temperature sensor 4 detects and displays the temperature inside the pressure vessel 1 and the pressure gauge 5 detects the pressure inside the pressure vessel 1.

  The pressurizing process is performed as follows, for example. First, a sealed container of a sample solution containing spores in a flexible container is placed inside the pressure-resistant container 1. Next, the pressure vessel 1 is filled with fresh water and subjected to pressurization with hydrostatic pressure. A container filled with spores can be made of various materials such as polyethylene and fluororesin. Moreover, as long as at least a part of the container to be filled has plasticity, the shape and the sealing method are not particularly limited.

The substance mixed in the spore in the substance mixing step according to the present embodiment is not particularly limited as long as it is a substance that penetrates into normal cells other than the spore. Examples of the substance include a compound containing a dye such as a fluorescent dye, a protein such as an antibody, an oligonucleotide, a plasmid, and a radioisotope. Non-radiolabeled ^ions such as K ⁺ and Ca ²⁺ are also included.

  Examples of fluorescent dyes include DAPI, Propium Iodide, Acidi Orange, SYBRGreen, Hoechst 33342, TOTO-1, CFDA, Calcein-AM, fluorescein, tetramethylrhodamine, rhodamine 6G, rhodamine green (trademark), rhodamine red (trademark), Oregon Green® 488, Texas Red®, Body Pie® (FL, R6G, TMR, TR and 630/650), Cy® (2, 3, 5 and 7), and Alexa Fluor® (350, 405, 430, 488, 532, 546, 555, 568, 594, 633, 647, 680, 700 and 750).

Proteins and oligonucleotides may be used as they are, or those labeled with the above dyes may be used. Examples of the oligonucleotide include probes used in the FISH method such as EUB338, and probes used in RT-PCR such as Taqman probe, Hyprobe, and Q primer. Examples of the plasmid include a plasmid having a gene encoding a labeled protein such as GFP. Examples of the radioisotope include tritium, ¹⁴ C, ⁴⁰ K, ¹³¹ I, and the like.

  A method for mixing the spore and the substance is not particularly limited. For example, a method of mixing the substance in a sample solution in which the spore is suspended in a buffer solution can be mentioned. In this case, the timing for adding the substance to the sample solution may be before the pressure treatment step or after the pressure treatment step. By adding the substance to the sample solution before the pressure treatment step, germination by pressurization and substance introduction into the spore can be performed simultaneously. In general, pressurization has the effect of impregnating a solute into an object. Therefore, if the substance is stable to pressurization and heating, it can be added to the sample solution by adding the substance to the sample solution before the pressurization process. The substance introduction effect can be enhanced. On the other hand, when the substance is unstable to pressurization and heating, it is preferable to add the substance to the sample solution after the pressurization process.

  In this embodiment, if only heat-resistant spores are targeted, heat shock treatment may be performed before the pressure treatment step or after the pressure treatment step.

  In the method for introducing a substance into a spore according to this embodiment, it is preferable to perform a pressure treatment step in the presence of a germination inducer. By applying the pressure treatment after adding the germination inducer, the effect of introducing the spore substance can be further improved. Examples of the germination inducer include amino acids such as alanine and asparagine, sugars such as glucose and fructose, and metal ions such as potassium. The concentration of the germination inducer is not particularly limited as long as it is a concentration that induces germination, and examples thereof include 10 mM. Moreover, since the substance introduction effect to a spore increases by using a germination inducer together, the pressure and time at the time of a pressurization process can be reduced.

  The method for introducing a substance into spores according to the present embodiment further includes an atmospheric pressure treatment step of holding the spores at a pressure of 5 ° C. to 70 ° C. for 1 to 60 minutes after the pressure treatment step. It is preferable to provide. The substance introduction effect of the spore can be further improved. This is because germination proceeds even if the spore whose metabolic pathway is activated by the pressure treatment returns to atmospheric pressure. In addition, the provision of the atmospheric pressure treatment step tends to reduce the treatment time in the method for introducing substances into spores according to the present embodiment. The atmospheric pressure treatment step is highly effective particularly when the pressure treatment step is performed in the presence of a germination inducer. In the atmospheric pressure treatment step, the holding temperature is 5 ° C to 70 ° C, the holding time is preferably 1 minute to 60 minutes, the holding temperature is 25 ° C to 50 ° C, and the holding time is 1 minute to 15 minutes. It is more preferable that Under these conditions, germination proceeds in a shorter time, and thus there is a tendency that the growth of germinated spores and microorganisms other than the spores can be further suppressed. Furthermore, it can suppress that the spore germinated by the temperature of 50 to 70 degreeC in a pressurization process process partially dies. When the holding time is less than 1 minute, the effect of introducing the spore substance tends to decrease. When the holding time exceeds 60 minutes, spores and microorganisms other than the spores tend to grow. If the holding temperature is less than 5 ° C., the germination promoting effect tends to decrease. When the holding temperature exceeds 70 ° C., spores with low heat resistance tend to be partially killed.

  As described above, according to the method for introducing a substance into a spore according to the present embodiment, by applying a specific pressure and temperature, a target substance can be quickly applied to a spore of a wide range of genera. It becomes possible to introduce efficiently.

  Next, a method for measuring spores having germination activity according to the present embodiment will be described. The method for measuring a spore having germination activity according to the present embodiment comprises a step of introducing a marker substance into a target spore by the substance introduction method into the spore.

  By providing such a process, it becomes possible to introduce a substance serving as a marker only to spores having germination activity, that is, living spores. As a result, it becomes possible to distinguish between living spores and dead spores in a short time. In addition, since pressurization has an action of inhibiting the growth of microorganisms, spores and other microorganisms do not grow in the process of introducing a marker substance into the spores. Therefore, measurement errors due to proliferation can be prevented. One type of marker may be used alone, or a plurality of types may be used in combination. When a plurality of types of markers are used, the number of spores may be determined using the difference in introduction efficiency due to germination of each marker.

  In the method for measuring spore having germination activity according to this embodiment, the conditions for the pressure treatment are the same as the conditions for the pressure treatment step in the method for introducing substances into the spore according to this embodiment, but the temperature is 5 More preferably, the temperature is from 50 ° C. to 50 ° C., and the pressure is from 10 MPa to 200 MPa. Under such conditions, there is less influence on microorganisms such as vegetative cell morphology that is less heat-resistant or pressure-resistant than spores, and there is a tendency that error factors in measuring the number of spores and the number of microorganisms other than spores are reduced. . On the other hand, a pressure of 200 MPa or more may have a bactericidal action on spores, but if a dye such as CFDA that develops color by esterase activity activated at the early stage of germination is used, the germinated spores are partially killed by the pressure. Therefore, if necessary, a pressure of 200 MPa or more may be applied.

  The timing of adding a substance that becomes a marker to the spore (substance mixing step) may be before the pressure treatment step or after the pressure treatment step, but after the pressure treatment step. Is preferred. There is a tendency that deterioration due to heating or pressurization of a substance serving as a marker can be suppressed. On the other hand, when measuring microorganisms other than a spore with a spore, it is preferable that the time which adds the substance used as a marker to a spore is before a pressurization process process.

  In the method for measuring spores having germination activity according to the present embodiment, the method for detecting spores having germination activity is not particularly limited. When the substance serving as the marker is a fluorescent dye, examples thereof include a flow cytometer method, a membrane filtration method, and a method using a CCD camera. Examples of the fluorescent dye that can be used include the dyes described above. In addition, by utilizing the improvement in permeability of fluorescent dyes by germination, the spores are fluorescently stained before and after the pressure treatment step, and the amount of increase in the staining rate before and after the pressure treatment step is compared and the bacteria are compared. There is also a method for obtaining a number. The method for measuring spore having germination activity according to the present embodiment may be a method other than fluorescent staining, such as a method using PCR, as long as a reagent is introduced into the spore.

  As mentioned above, although embodiment of this invention was described, this invention is not limited to the said embodiment. For example, the method for introducing a substance into a spore according to the present invention comprises analyzing the intracellular structure of a spore by introducing a compound containing a radioisotope and a fluorescent dye, modifying the spore by introducing a plasmid, and detecting the spore by PCR. And can be used for quantification.

  The invention will now be described in more detail by the following examples. However, these examples do not limit the present invention.

Study 1: Effect of pressure treatment on rapid measurement of spore count (pressure condition)
B. Table 1 shows the results of calculating the number of spores by fluorescent staining after pressurizing subtilis (IAM 12118) spores under conditions of 0.1 to 400 MPa, 40 ° C., and 15 minutes. That is, B. so as to have a concentration of 10 ⁶ CFU / ml. Subtilis (IAM 12118) spores were suspended in 1/15 M phosphate buffer (pH 7.0) and aseptically sealed and filled in polyethylene bags. A polyethylene bag filled with the spore suspension was placed in a pressure heating apparatus (manufactured by Hikari Kouhiki Co., Ltd.) shown in FIG. The spore suspension subjected to the pressure treatment was aseptically transferred to the reaction vessel. Subsequently, 6-Carboxyfluorescein diacetate: CFDA (Sigma-Aldrich) solution was added to the reaction vessel so as to have a concentration of 150 μg / ml, and the germinated spores were stained by reacting at 25 ° C. for 10 minutes. The stained spore suspension was filtered through a polycarbonate membrane filter (Advantech Toyo Co., Ltd.) having a diameter of 13 mm and a pore diameter of 0.2 μm. The fluorescently stained spores on the membrane were observed with an epifluorescence microscope (manufactured by Olympus Corporation) at a magnification of 1000 times. Twenty visual fields were randomly selected from the observed visual fields, and fluorescently stained spores were counted. The number of spores in the fluorescent staining method was calculated according to the following formula.
Number of fluorescently stained spores (cells / mL) = (average number of fluorescently stained spores per field) × (filtration area of membrane filter) / (area per field) / (hang of stained spores) Filtration amount of turbid liquid) ... [1]
In addition, the spore count of the spore suspension used in the fluorescence staining method was also determined by the culture method and compared with the result of the fluorescence staining method. The culture method was performed by the following method. That is, the spore suspension was heated at 70 ° C. for 20 minutes, diluted appropriately, and cultured on a pour plate using a standard agar medium (manufactured by Nissui Pharmaceutical Co., Ltd.) at 35 ° C. for 48 hours. The formed colonies were counted. The number of spores in the culture method was calculated according to the following formula.
Spore count (CFU / mL) = (average colony count) × (dilution factor) [2]

  As shown in Table 1, it was found that spores could be germinated by pressure treatment of 10 to 400 MPa, particularly 50 to 400 MPa, and then the number of spores could be measured by fluorescent staining. On the other hand, those treated with 0.1 MPa did not stain spores, and the number of spores could not be measured. Note that the number of spores did not change much even at a pressure of 200 MPa or more.

Study 2: Effect of pressure treatment on rapid measurement of spore count (temperature conditions)
B. Spores stained by the same method as in Study 1 were counted except that the spores of subtilis (IAM 12118) were subjected to pressure treatment under conditions of 100 MPa, 10 to 70 ° C., and 15 minutes. In addition, the number of spores was determined by the same culture method as in Study 1. These results are shown in Table 2. In addition, the number of spores by the fluorescent staining method and the culture method was calculated by the above formula [1] and formula [2].

  As shown in Table 2, it was found that spores can be efficiently germinated at a temperature of 5 to 70 ° C., particularly 25 to 55 ° C., and then the number of spores can be measured by a fluorescent staining method.

Study 3: Effect of pressure treatment on rapid measurement of spore count (treatment time)
B. Spores stained in the same manner as in Study 1 were counted except that the spores of subtilis (IAM 12118) were subjected to pressure treatment under conditions of 100 MPa, 40 ° C., and 1 to 60 minutes. In addition, the number of spores was determined by the same culture method as in Study 1. These results are shown in Table 3. In addition, the number of spores by the fluorescent staining method and the culture method was calculated by the above formula [1] and formula [2].

  As shown in Table 3, it was found that spores could be germinated in a treatment time of 1 to 60 minutes, and then the number of spores could be measured by fluorescent staining.

Study 4: Effect of pressure treatment on rapid measurement of spores of various genus species Except that spores of various genus species were used as spores and pressure treatment was performed at 100 MPa, 40 ° C. for 15 minutes. Spores stained by the same method as in Study 1 were counted. In addition, the number of spores was determined by the same culture method as in Study 1. These results are shown in Table 4. In addition, the number of spores by the fluorescent staining method and the culture method was calculated by the above formula [1] and formula [2].

  As shown in Table 4, it was found that the number of spores can be measured in a short time by the fluorescent staining method for spores of a wide range of genus species by pressure treatment.

Study 5: Combined effect of germination inducer Spores isolated from food (gene identification result by 16sRDNA: Bacillus pumilus) in 1/15 M phosphate buffer (pH 7.0) to a concentration of about 10 ⁶ CFU / ml After suspending and adding L-Ala to 10 mM, pressure treatment was performed under conditions of 50 MPa, 40 ° C., and 5 minutes. Thereafter, the stained spores were counted by fluorescent staining. Further, L-Ala was added to the spore suspension so as to be 10 mM, and the mixture was treated under atmospheric pressure (0.1 MPa) at 40 ° C. for 15 minutes. In addition, the number of spores was determined by the same culture method as in Study 1. Thereafter, the stained spores were counted by fluorescent staining. These results are shown in Table 5. In addition, the number of spores by the fluorescent staining method and the culture method was calculated by the above formula [1] and formula [2].

  As shown in Table 5, it was found that by adding L-Ala, which is a germination inducer, the effect of introducing a fluorescent dye by pressure treatment can be further enhanced, and the measured value of the number of spores can be made more accurate. In addition, it was found that by performing the pressure treatment in the presence of the germination inducer, the pressure in the pressure treatment can be reduced and the treatment time can be shortened.

Spores separated from food (gene identification result by 16sRDNA: Bacillus pumilus) are suspended in 1 / 15M phosphate buffer (pH 7.0) to a concentration of about 10 ⁶ CFU / ml, and L-Ala is made 10 mM. After the addition, pressure treatment was performed under the conditions of 50 MPa, 40 ° C., and 5 minutes. A part of the suspension subjected to the pressure treatment was kept under atmospheric pressure at 40 ° C. for 30 minutes. Thereafter, the stained spores were counted by fluorescent staining. In addition, the number of spores was determined by the same culture method as in Study 1. These results are shown in Table 6.

  As shown in Table 6, it was found that the effect of introducing the fluorescent reagent can be further enhanced by maintaining the pressure under atmospheric pressure after the pressure treatment. Further, it was found that by adding a step of maintaining under atmospheric pressure (atmospheric pressure treatment step), the pressure in the pressure treatment can be reduced and the treatment time can be shortened.

  DESCRIPTION OF SYMBOLS 1 ... Pressure-resistant container, 2 ... Heater, 3 ... Pressure pump, 4 ... Temperature sensor, 5 ... Pressure gauge.

Claims (7)

A pressure treatment step of pressurizing the target spores at a temperature of 5 ° C. or more and 70 ° C. or less for 1 second or more and 60 minutes or less, 10 MPa or more and 100 MPa or less;
A substance mixing step of mixing the spore and a substance serving as a marker to be introduced into the spore;
An atmospheric pressure treatment step in which the spore introduced with the substance serving as a marker is held at a temperature of 25 ° C. or more and 70 ° C. or less at atmospheric pressure for 1 minute or more and 60 minutes or less;
Detecting the spore introduced with the substance to be a marker by detecting the substance to be a marker; and
With
The method for measuring spores having germination activity, wherein the substance mixing step may be before or after the pressure treatment step.   The measurement method according to claim 1, wherein the pressure treatment step is performed in the presence of a germination inducer.   The measurement method according to claim 2, wherein the germination inducer is an amino acid, a sugar, or a metal ion.   The measurement method according to claim 2 or 3, wherein the germination inducer is an amino acid. The substance serving as marker, a color element that develops color by esterase activity, measuring method according to any one of claims 1-4. The measurement method according to any one of claims 1 to 5, wherein the substance serving as a marker is 6-Carboxyfluorescein diacetate (CFDA) .   The measurement method according to any one of claims 1 to 6, wherein the substance mixing step is performed before the pressure treatment step.

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