WO2013051496A1 - Method for assessing cell heat resistance by determining temperature dependence of cell shape or hardness - Google Patents

Method for assessing cell heat resistance by determining temperature dependence of cell shape or hardness Download PDF

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WO2013051496A1
WO2013051496A1 PCT/JP2012/075345 JP2012075345W WO2013051496A1 WO 2013051496 A1 WO2013051496 A1 WO 2013051496A1 JP 2012075345 W JP2012075345 W JP 2012075345W WO 2013051496 A1 WO2013051496 A1 WO 2013051496A1
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cell
temperature
value
cells
heat resistance
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PCT/JP2012/075345
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French (fr)
Japanese (ja)
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弘一 中西
亮雅 小暮
良平 粉川
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株式会社島津製作所
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • C12Q1/06—Quantitative determination

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  • the present invention relates to a method for evaluating the heat resistance of cells, and more specifically, to a method for evaluating the heat resistance of cells by determining the temperature dependence of the shape or hardness of the cells.
  • Patent Document 1 a method for evaluating the heat resistance of a cell by measuring the hardness of the cell.
  • This method is a method for evaluating the heat resistance of cells utilizing the fact that the hardness of cells and the heat resistance of cells are proportional. That is, by applying the hardness at a specific temperature of a cell of unknown heat resistance to a calibration curve prepared in advance with respect to the hardness and heat resistance of the cell using various cells, the heat resistance of the cell at a specific temperature can be evaluated. it can. According to this method, as long as a calibration curve is prepared, the heat resistance of the cells at a specific temperature can be easily and rapidly evaluated thereafter.
  • the evaluation object is limited to spores of spore-forming bacteria in which a proportional relationship between cell hardness and cell heat resistance is recognized.
  • thermophilic obligate anaerobic spore-forming bacteria spores and mold ascospores there is no correlation between cell hardness and heat resistance, and it is not applicable to such microbial species. there were. Therefore, development of a simple and rapid method for evaluating the heat resistance of cells applicable to a wide range of microbial species has been desired.
  • An object of this invention is to provide the method of evaluating the heat resistance of a cell simply and rapidly.
  • the present inventors have found that the heat resistance of cells can be easily and rapidly evaluated by determining the temperature dependence of the cell shape or hardness.
  • the present invention is based on such knowledge.
  • a method for evaluating the heat resistance of a cell comprising a step of determining the temperature dependence of the shape or hardness of the cell.
  • SPM scanning probe microscope
  • the heat resistance of the cell can be evaluated easily and quickly. Since cell isolation is not required, the cell mixture can be used for measurement as it is. Therefore, heat resistance in the presence environment of cells can be evaluated. Moreover, according to this invention, D value in arbitrary heating temperature or the heating temperature which shows arbitrary D values can be evaluated simply and rapidly. Therefore, according to the present invention, for example, the heat resistance in the presence of bacteria can be evaluated, and the heat treatment time of the food or drink can be determined according to the current situation.
  • FIG. 1 is a diagram showing the relationship between the cell heating temperature of the measurement sample prepared from the test microorganism strain and the increase in cell thickness at that time.
  • the horizontal axis is the cell heating temperature (° C.), and the vertical axis is the amount of increase in cell thickness (nm) from 25 ° C.
  • FIG. 1A shows results in spore-forming bacteria spores ( B. licheniformis and B. coagulans )
  • FIG. 1B shows results in thermophilic obligate anaerobic spore-forming bacteria ( T. mathranii and M. thermoacetica )
  • FIG. 1C shows results in normal cells (vegetative cells) ( E. coli , S. aureus subsp. Aureus and S. pastrianus )
  • FIG. 1A shows results in spore-forming bacteria spores ( B. licheniformis and B. coagulans )
  • FIG. 1B shows results in thermophilic obligate anaerobic spore
  • FIG. 1D shows results in mold ascospores ( B. fulva and T. flavus ). Indicates.
  • the vertical line in the graph is a straight line for determining the cell-specific temperature T 1 or T 2 .
  • FIG. 2 is a diagram showing a typical heat-killing time curve (TDT curve) of microorganisms. The data is shown on a semilogarithmic graph in which the vertical axis is the heating temperature (° C.) and the horizontal axis having a logarithmic scale is the D value (minutes).
  • ⁇ shows the relationship between the heating temperature and the D value in the B. licheniformis strain
  • ⁇ shows the relationship between the heating temperature and the D value in the B. coagulans strain.
  • FIG. 3 is a diagram showing that the temperature of T 1 changes by changing the heating rate of the cantilever during the thermal analysis of the cell thickness.
  • the relationship between cell thickness and heating temperature in B. licheniformis and B. coagulans at a rate of 10 ° C per second or 20 ° C per second is shown in the graph, and the temperature of T 1 changes depending on the rate of temperature increase. I understand.
  • Examples of cells to be evaluated in the present invention include prokaryotes such as bacteria, eukaryotes such as fungi, and archaeal cells. Any cell can be used as a measurement target as long as it is a unicellular organism. Even cells of multicellular organisms such as animals and plants can be used for measurement. In the case of a unicellular organism, it can be measured with or without a cell wall. Even if there is a cell wall, it can be measured regardless of the type of the cell wall, and it is not necessary to remove the cell wall or the like in the measurement. Therefore, even for Gram-negative bacteria, fungi, plants, and archaea cells having a thick cell wall, the hardness or shape of the cells can be measured by a simple procedure. In the present specification, “cell” may be used to mean a unicellular organism itself. *
  • the bacterium to be measured in the present invention is not particularly limited as long as it is a bacterium.
  • Measurement targets include Staphylococcus, Listeria, Lactobacillus, Bifidobacterium, Enterococcus, Lactococcus, Pediococcus, Leuconostoc, Streptococcus, Vibrio, Salmonella, Escherichia Bacteria of the genus Campylobacter, Amphibacillus, Bacillus, Geobacillus, Clostridium, Desulfotomacrum, Thermoanaerobacter, Murella and Sporosarcina, preferably Staphylococcus aureus; Listeria monocytogenes; Lactobacillus; Vibrio parahaemolyticus; Salmonella; Escherichia coli; Campylobacter; and Amphibacillus, Bacillus, Geobacillus, Clostridium, Desulfotomacrum, Thermoanaerobacter, Muellera and Sporosarcina Spore-forming bacterium is more preferable.
  • Staphylococcus aureus Staphylococcus aureus Staphylococcus aureus, in particular Staphylococcus aureus subsp. Aureus, E. coli Escherichia coli, and a spore-forming bacterium Bacillus Bacillus subtilis, Bacillus subtilis ver. Subtilis , Bacillus coagulans, Bacillus licheniformis, Bacillus megaterium, spore-forming bacteria Geobacillus sp Most preferred are Geobacillus stearothermophillus , Thermoanaerobacter mathranii , Moorella thermoacetica , which are thermophilic obligately anaerobic spore-forming bacteria.
  • the fungi to be measured in the present invention include Basidiomycota, Ascomycota, Chytridiomycota, Neocallimastigomycota, Blastocladiomycota, microspore Examples include fungi such as Microsporidia, Glomeromycota, Mucoromycotina, Haechobi Entomophthoromycotina, Zoopagomycotina, Kickxellmycotina.
  • yeast belonging to the Basidiomycota or Ascomycota or fungus (mold) belonging to the zygomycota, Ascomycota or Basidiomycota is preferred.
  • yeast examples include, for example, the genus Saccharomyces, Candida, Torulopsis, Zygosaccharomyces, Schizosaccharomyces, Pichia, Yia, Yeasts belonging to the Ascomycota such as Hansenula, Kluyeromyces, Debaryomyces, Geotrichum, Wickerhamia and Fellomyces Yeast belonging to basidiomycetes such as Sporobolomyces is preferred. More preferred are Saccharomyces cerevisiae and Saccharomyces pastrianus which are budding yeast of the genus Saccharomyces , and Schizosaccharomyces pombe which is a fission yeast of the genus Schizosaccharomyces .
  • the fungi to be measured are preferably fungi belonging to Ascomycota such as Byssochlamys and Talaromyces, and more preferably Byssochlamys fulva and Talaromyces flavus . These Ascomycota fungi are subject to measurement in the present invention as they are even in the state of ascospores.
  • the cells to be measured in the present invention not only isolated and cultured cells, but also cells or a mixture of cells mixed in foods and drinks, solutions, and the like can be used. This is because the cells to be measured according to the present invention can be collected directly from the presence environment of the cells and used for direct measurement. In the present invention, if at least one cell can be measured, the heat resistance of the cell can be evaluated. Therefore, it is not always necessary to isolate the cell to be evaluated, and the heat resistance of the cell in the environment where the cell exists is evaluated. Is possible.
  • the cells to be measured according to the present invention can be used for measurement after being cultured under culture conditions suitable for the growth of the cells. Those skilled in the art can appropriately determine the culture conditions suitable for the growth of each cell.
  • the cells are collected directly from the environment in which the cells exist or are used for measurement, or cultured under culture conditions suitable for the growth of the cells and then used for measurement, the cells are appropriately fixed and used for measurement. be able to. Although it does not specifically limit as a fixing method which measures a living cell, Air dry fixation is mentioned. *
  • the heat resistance of cells can be evaluated by determining the temperature dependence of the shape or hardness of the cells. As shown in the examples below, when cells with constant stress are heated at a constant rate of temperature increase, the shape of the cells changes in a temperature-dependent manner, and the rate of change rapidly changes at a certain temperature. Furthermore, it has been found that the temperature is an indicator of the heat resistance of the cells. Therefore, according to the present invention, the temperature dependence of the cell shape (for example, an index that can objectively identify a change in cell shape such as cell thickness or diameter (length and / or width in the case of Neisseria gonorrhoeae)). By determining the sex, the heat resistance of the cells can be evaluated.
  • the heat resistance of the cell may be evaluated by determining the temperature dependency of the hardness of the cell, and further, the temperature dependency of the physical property value of the cell such as Young's modulus and elastic modulus is determined. You may evaluate the heat resistance of a cell by determining. Therefore, according to the present invention, the heat resistance of a cell is evaluated by determining the temperature dependency of the cell shape or the temperature dependency of the physical property value of the cell such as the hardness, Young's modulus or elastic modulus of the cell. Can do. Although not bound by the following theory, when cells are placed under elevated temperature, the physical properties of the cells themselves change before and after cell death, and the heat resistance of the cells is evaluated by capturing the changes. Will be possible. *
  • the step of determining the temperature dependence of the cell shape may be performed in a state where stress is applied to the cell, or may be measured without applying stress.
  • stress it is not particularly limited, but the shape of the cell in the direction in which stress is applied can be measured.
  • the cell shape is cell thickness.
  • An apparatus for measuring a change in cell shape can be used without particular limitation as long as the cell shape can be measured, and examples thereof include a scanning probe microscope (SPM).
  • SPM scanning probe microscope
  • AFM atomic force microscope
  • a method for measuring the shape of a cell using a scanning probe microscope is well known to those skilled in the art. *
  • the stress on the cells can be generated using a cantilever such as a scanning probe microscope.
  • the shape of the cell can be measured using a cantilever such as a scanning probe microscope.
  • the cantilever for stress generation and cell shape measurement can be separate cantilevers, but preferably the same cantilever is used to generate stress and simultaneously measure the cell shape at that time Can do. *
  • the hardness of the cell can be represented by an elastic modulus or Young's modulus (N / m 2 or Pa). Therefore, the step of determining the temperature dependency of the cell hardness can be a step of determining the temperature dependency of the elastic modulus or Young's modulus of the cell. That is, the temperature dependence of cell hardness can be determined by measuring the elastic modulus or Young's modulus of cells at various temperatures. Note that the elastic modulus and Young's modulus are common in that they are physical property values representing the difficulty of deformation, and therefore both are treated as synonymous in this specification.
  • Young's modulus E is generally Can be expressed as Here, the stress ⁇ is a force per unit area (N / m 2 or Pa), and the strain ⁇ is the amount of expansion / contraction per unit length of deformation due to the stress ⁇ . Therefore, the Young's modulus E of the cell can be determined from the stress ⁇ applied to the cell and the strain ⁇ of the cell based on the above formula.
  • the cell strain ⁇ can be obtained, for example, by dividing the cell thickness when the stress ⁇ is loaded by the cell thickness when no load is applied.
  • the cell strain ⁇ (i) the amount of change in the cell thickness or thickness when stress ⁇ is applied, or ( ii) The temperature dependence of the Young's modulus of the cell can be determined using the stress ⁇ required for making the cell thickness constant.
  • the cell strain ⁇ may be determined not only by the cell thickness but also by the cell diameter (length and / or width in the case of Neisseria gonorrhoeae).
  • the temperature dependence of cell shape or hardness can be determined by measuring these while raising the temperature of the cells.
  • the means for raising the temperature of the cells is not particularly limited.
  • the temperature can be raised by a sample stage or cantilever brought into contact with the cells, or the temperature can be raised by placing the sample in a heated atmosphere.
  • the temperature increase can be performed preferably with a constant temperature change (temperature increase rate) per unit time.
  • the rate of temperature rise is not limited, but can be, for example, 0.1 ° C. per second to 100 ° C. per second, 1 ° C. per second to 50 ° C. per second, or 10 to 20 ° C. per second. it can.
  • the temperature rise of the cell and the measurement of the shape or hardness can be performed by independent means, but preferably the temperature rise and the measurement of the shape or hardness can be performed simultaneously using a cantilever equipped with a heating means. .
  • a cantilever provided with a heating means for example, a local heating type cantilever can be mentioned, and an SPM provided with a local heating type cantilever can be preferably used.
  • the temperature at which the temperature dependence of the cell shape or hardness characteristically changes can be determined. That is, in the method for evaluating the heat resistance of cells according to the present invention, a) In addition to the step of determining the temperature dependence of cell shape or hardness, b) the temperature dependence from the temperature dependence of the determined shape or hardness. The method may further include a step of deriving a temperature at which is characteristically changed. The temperature at which the temperature dependency obtained in step b) is characteristically changed can be used for comparison of the heat resistance of cells by comparing the levels. The temperature at which such temperature dependence changes characteristically can be derived as follows. *
  • the temperature at which the temperature dependence of the cell shape or hardness changes is the cell shape (for example, thickness), or the cell shape or cell shape on a graph in which the reciprocal of the cell hardness is plotted against the heating temperature.
  • the temperature at which the slope of the reciprocal hardness changes abruptly (sometimes referred to simply as “T 1 ” (° C.) in the present specification) or the shape of the cell, or the temperature at which the reciprocal of the hardness of the cell exhibits a maximum value ( In the present specification, “T 2 ” (sometimes referred to as “° C.”) is applicable.
  • the temperature T 1 (° C.) may be obtained visually from the graph, or two points are arbitrarily selected from before and after the visually obtained T 1 , and the distance from the straight line passing through the two points is the longest. You may obtain
  • the heating temperature T 2 (° C.) at which the reciprocal of the cell shape or the cell hardness has a maximum value can be obtained, for example, as the temperature at which the reciprocal of the cell shape or the cell hardness shows the maximum value.
  • Examples of such cells having T 2 include vegetative cells of spore-forming bacteria, bacteria other than spore-forming bacteria, and fungi. In these cells, since the data shows high linearity before and after the temperature T 2 , the shape or the reciprocal of hardness is obtained from two regression curves or a line regression model in the same manner as when the temperature T 1 is determined.
  • the heating temperature T 2 (° C.) at which can be a maximum value may be obtained.
  • the step of deriving the cell-specific temperature T 1 or T 2 from the temperature dependence of the determined cell shape or hardness can also be performed by a method mathematically equivalent to the above.
  • a mathematically equivalent method is to derive the temperature T 1 or T 2 by reversing the measured value, multiplying the measured value by a constant (or adding a certain constant), or dividing by a certain constant.
  • a method of deriving the temperature T 1 or T 2 by combining (or subtracting a certain constant) or combining these additions, subtractions, multiplications and divisions can be mentioned.
  • the temperature at which the reciprocal of cell shape or cell hardness shows the maximum value is determined as the temperature at which the reciprocal of cell shape or hardness shows the minimum value (minimum value). But you can ask.
  • the temperature of T 1 or T 2 thus determined can be used for evaluating the heat resistance of each microorganism by comparing the temperature. Specifically, T 1 or T 2 of various microorganisms are compared, and it can be evaluated that the higher the temperature of T 1 or T 2 , the higher the heat resistance.
  • the time required to reduce the survival number of microorganisms to one-tenth at a certain temperature (referred to simply as “D value” in the present specification). And a heating temperature showing a certain D value can also be obtained.
  • a temperature at which the temperature dependence changes characteristically may include a step of determining a corresponding D value, and in addition to step c), d) a step of calculating a heating temperature indicating a desired D value or a D value at a desired heating temperature. Further, it may be included. Furthermore, the heat sterilization conditions of microorganisms can be determined from the heating temperature indicating the desired D value obtained in step d) or the D value at the desired temperature. In addition, as a general heat sterilization theory, the theory described in “First-hand collection of practical data on microorganisms / microbe sterilization”, first edition, pages 31 to 42 can be mentioned.
  • the step c) of determining the D value corresponding to the cell-specific temperature T 1 or T 2 can be performed as follows. Specifically, the value of the D value corresponding to T 1 or T 2 determined by the method of the present invention can be easily determined by those skilled in the art by using a general microorganism heat sterilization theory. Can be sought.
  • the corresponding D value takes a substantially constant value regardless of the bacterial species. That is, when the step a) is performed with the temperature rising rate condition being constant, and then the step b) is performed, the temperature T 1 or T 2 determined thereby corresponds to a specific D value regardless of the bacterial species. Will do. According to this knowledge, the heating temperature T and the number of surviving microorganisms are reduced to 1/10 for a certain microorganism (preferably 2 to 3 kinds of microorganisms) by a general method based on the heat sterilization theory of microorganisms.
  • the relational expression for the required time D is determined.
  • the temperature T 1 or T 2 determined in the step a) and the step b) performed at a specific heating rate condition for the microorganism is introduced into the determined relational expression, and the corresponding D value is once determined. Then, it can be determined that the temperature T 1 or T 2 determined for other microorganisms thereafter has the same D value as long as the temperature T 1 or T 2 is measured using the same heating rate condition. Since the D value determined as described above changes depending on the temperature increase rate of the cells, when changing the temperature increase rate condition in step a), determine the D value for each temperature increase rate condition. Is preferred.
  • the TDT curve is determined for each environment where the microorganism to be measured exists, and based on the determined TDT curve, the temperature at which the temperature dependence changes characteristically, that is, D corresponding to the cell-specific temperature T 1 or T 2. The value may be determined. In this case, it may be determined as D value corresponding to the temperatures T 1 or T 2 of the under the circumstances (culture conditions).
  • the regression equation of the TDT curve can be obtained by the following two methods: (i) a method of determining the regression equation of the TDT curve from a temperature indicating a certain D value and a constant d; or (ii) a certain D value.
  • a method of determining a regression equation of a TDT curve from a temperature indicating the temperature and a temperature indicating another D value By using the regression equation thus determined, it is possible to calculate a D value at an arbitrary heating temperature or a heating temperature indicating an arbitrary D value, and further, a heating temperature or a desired temperature indicating a desired D value.
  • the D value at the heating temperature can be calculated.
  • the desired D value is obtained by measuring the cells under the heating rate condition. You may calculate the heating temperature which shows a value.
  • the regression equation can be determined as follows.
  • the value corresponds to a change in heating temperature (° C.) corresponding to a change of 10 times or 1/10 of the D value. Since it is possible for those skilled in the art to calculate the Z value (or constant d) from the already known D value under normal culture conditions, in step c) a specific T 1 or T 2 and its corresponding D If even one value can be calculated, the regression equation of the TDT curve can be estimated.
  • the D value under such known normal culture conditions can be obtained from, for example, a TKDB database (for example, ver3.0) provided by TriBiox Laboratories. Since the D value and Z value may fluctuate due to changes in the cell culture environment, it is preferable to use data obtained in an environment as close as possible to the environment in which the microorganism to be measured exists. Alternatively, the Z value may be corrected as appropriate to estimate the Z value in the presence of microorganisms, and used to derive a regression equation for the TDT curve.
  • TKDB database for example, ver3.0
  • the Z value may be corrected as appropriate to estimate the Z value in the presence of microorganisms, and used to derive a regression equation for the TDT curve.
  • the regression equation can be determined as follows.
  • the regression equation of the TDT curve can be obtained from the cell-specific temperature T 1 or T 2 obtained under two or more different temperature raising conditions and the corresponding D value. At this time, one or more of the two or more D values can be substituted with the D value of the TKDB database.
  • the method (ii) is advantageous in that a regression equation of a TDT curve can be accurately obtained in the presence environment (culture environment) of microorganisms.
  • the actual heat sterilization conditions can be determined using the D value at an arbitrary temperature obtained by the method of the present invention. For example, by setting the heating time to 5D or 12D, the number of surviving microorganisms can be reduced on the order of 10 5 or 10 12 , respectively.
  • the heat sterilization time can be 5D.
  • the heat sterilization time can be set to 12D.
  • the heat sterilization time can be set to 6D.
  • Such a heat sterilization time can be appropriately set by those skilled in the art in consideration of the toxicity of bacteria to be sterilized and the preservability of food and drink to be sterilized.
  • Example 1 Construction of a scanning probe microscope (SPM) having a heating function
  • SPM scanning probe microscope
  • a nanosearch microscope in which a cantilever is replaced with a locally heated cantilever (manufactured by Nippon Thermal Consulting) as an SPM having a local heating function.
  • SFT-3500 manufactured by Shimadzu Corporation was used.
  • This cantilever has a sufficient temperature raising capability to raise the temperature to 200 ° C. or 400 ° C. at least at 20 ° C. while applying an arbitrary constant force of several tens of N to 100 N to the sample surface. It was. In the following examples, from the room temperature (25 ° C.) to 200 ° C.
  • thermophilic anaerobic anaerobic condition at a rate of temperature increase of 20 ° C. per second while applying a load of several tens of N to about 100 N on the center of the cell surface by a cantilever.
  • the cells were heated up to 400 ° C.), and the change (nm) in cell thickness at that time was measured.
  • the cell thickness or the change in the cell thickness can be calculated by a method well known to those skilled in the art with reference to an instruction manual attached to the SPM.
  • test microorganism strain sample 2-1 Test microorganism strains
  • test microorganism strains a wide variety of species such as spore-forming bacteria (spore and nutrient phase) of the genus Bacillus, thermophilic obligately anaerobic spore-forming bacteria, bacteria, fungi, and yeast were used.
  • Geobacillus stearothermophillus NBRC13737 strain Bacillus coagulans DSM1 strain, Bacillus subtilis NBRC13719T strain, Bacillus licheniformis NBRC12200 strain and Bacillus megaterium NBRC15308T strain were used as the spore-forming bacterium of the genus Bacillus.
  • Yeast Saccharomyces pastorianus RIB2010 strain was cultured at 25 ° C. for 48 hours using YM medium (Difco, product number: 271120). Thermophilic obligate anaerobic spore-forming bacteria were cultured at 55 ° C. for 72 to 96 hours using a modified TGC medium (manufactured by Nissui Pharmaceutical Co., Ltd., product number: 30206293). Other test microorganism strains were cultured at 35 ° C. for 24 hours using a gravy medium (Difco, product number: 234000).
  • thermophilic obligate anaerobic spore-forming bacteria anaerobic culture was performed using an anaerobic gas generating reagent Aneropack (registered trademark) and a gas generating agent for anaerobic culture (manufactured by Mitsubishi Gas Chemical Company).
  • yeast and bacteria measurement samples were prepared by collecting cells by centrifugation (5,000 rpm) and washing twice with pure water.
  • a sample for measuring spores was prepared by the method described on pages 19 to 30 of “Spore Experiment Manual” edited by Masaomi Kondo and Kazuhito Watanabe, published by Gihodo Publishing (1995). Specifically, after spore-forming bacteria were grown in a 50 mL broth medium for 48 hours at 35 ° C. and grown, 100 ⁇ g / mL lysozyme (Wako Pure Chemical Industries) was added to the cells recovered by centrifugation (5,000 rpm).
  • a sample for measuring mold ascospores was prepared by the method described on pages 130-131 of Kosuke Takashima, mold inspection manual color chart, Techno System (2002). Specifically, using a potato dextrose medium (manufactured by Nissui Pharmaceutical Co., Ltd., product number: 302057092), mold was cultured at 25 ° C. for 1 month, and ascospore formation was confirmed under an optical microscope. A sample for measuring mold ascospores (ascospore sample) was used.
  • a sample of the cell or spore suspension thus prepared was dropped onto a slide glass and fixed by air drying.
  • the sample after air drying fixation was used for measurement immediately.
  • the cells after air-drying fixation are considered to be alive.
  • Example 3 Measurement of increase in thickness (nm) of sample cell for measurement by thermal analysis SPM locally heated cantilever is brought into contact with the center surface of the measurement sample cell fixed by air-drying on a slide glass, and the thickness direction of the cell A constant force (about several tens of N to 100 N) was continuously applied. In this state, while increasing the tip temperature of the cantilever to 200 ° C. at a constant rate (20 ° C. per second) (up to 400 ° C. in the case of thermophilic obligate anaerobic spore-forming bacteria), the increase in cell thickness (nm ) was monitored.
  • FIG. 1A shows the relationship between cell thickness (nm) and heating temperature (° C.) in spore samples ( B. licheniformis and B. coagulans ) among samples prepared from the test microorganism strains.
  • the increase in cell thickness (nm) tended to show a monotonous increase with increasing heating temperature (FIG. 1A).
  • T 1 ° C.
  • FIG. 1D shows the relationship between the increase in cell thickness (nm) and the heating temperature (° C.) in mold ascospore samples ( T. flavus and B. fulva ) among samples prepared from the test microorganism strains.
  • the graph of the increase in cell thickness (nm) with respect to the heating temperature tends to show a mountain shape, and the increase in cell thickness (nm) is the maximum value (maximum value). It was found that there was a temperature T 2 (° C.) at which the temperature falls (FIG. 1D downward arrow).
  • the relationship between the amount of increase in cell thickness (nm) and the heating temperature (° C.) varies greatly with the temperature T 1 or T 2 as a boundary. At such a temperature T 1 or T 2 , the physical properties of the cells are considered to change greatly.
  • Example 4 Relationship between Increase in Cell Thickness (nm) and Cell Death Before and after temperature T 1 or T 2 , the physiological state of the cell increases with the change in cell physical properties. It seems to have changed.
  • the general theory of heat sterilization for example, “In-Field Essential / Microbial Sterilization Practical Data Collection”, 1st edition, pages 31-42 According to the theory
  • the relationship between the D value in the heat sterilization theory and the temperature T 1 or T 2 was investigated.
  • thermal analysis of the increase in cell thickness (nm) can be a useful tool for simple and rapid evaluation of heat resistance for a wide range of microbial species.
  • the change in the thickness of the cell under a constant stress load is monitored.
  • the hardness of the cell, the Young's modulus, or the diameter, length, or width of the cell, or a change thereof is temporarily monitored. However, it is considered that the same result can be obtained.
  • Example 5 Characteristic temperatures T 1 and T 2 of the D value calculated in the thermal analysis and in order to investigate the relationship between the D value in more detail, the temperature rise rate of the cantilever is changed to change the temperature T 1 or T 2 (° C.) was determined, and the relationship with the D value was examined. First, it was confirmed that even if the rate of temperature increase was changed, T 1 or T 2 (° C.) could be obtained from the relationship between the temperature increase and the amount of increase in cell thickness (nm) (FIG. 3). Next, when the D value corresponding to the temperature T 1 or T 2 obtained from the regression equation of the TDT curve was calculated, it was found that the D value was 0.01 (min) (FIG. 2).
  • microorganisms have a heating time D value in which the number of cells becomes 1/10 when heated at a constant temperature, and the D value is an indicator of the heat resistance of the microorganism.
  • the heating time and the heating temperature for obtaining the same heat sterilization effect have a relationship that if one is increased, the other is decreased.
  • the temperature T is reached in a shorter heating time (where T is an arbitrary temperature) as the heating rate is increased.
  • T is an arbitrary temperature
  • the temperature T 1 or T 2 is considered to rise in a measurement environment having a high temperature rising rate.
  • the D value decreases. This is considered to be the reason why the temperature T 1 or T 2 (° C.) increases and the D value decreases when the rate of temperature increase is increased.
  • the rate of temperature increase is slowed down, the temperature T is not reached unless a longer time is taken.

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Abstract

Provided is a method for easily and rapidly assessing cell heat resistance. Cell heat resistance is assessed by determining the temperature dependence of cell shape or hardness. In one embodiment, a scanning probe microscope provided with a heating means is used for said assessment. Said assessment may also include a step for calculating the heating temperature of cells, indicative of a predetermined D value, or a step for calculating the D value of cells at a predetermined heating temperature.

Description

細胞の形状または硬度の温度依存性を決定することによる、細胞の耐熱性評価法Evaluation method of heat resistance of cells by determining temperature dependence of cell shape or hardness
本発明は、細胞の耐熱性評価法に関し、具体的には、細胞の形状または硬度の温度依存性を決定することによる、細胞の耐熱性評価法に関する。 The present invention relates to a method for evaluating the heat resistance of cells, and more specifically, to a method for evaluating the heat resistance of cells by determining the temperature dependence of the shape or hardness of the cells.
従来、細胞の耐熱性評価は、一般的な加熱殺菌理論に基づいて、試料を一定時間加熱し、加熱後の生残菌数を測定することにより行われていた。これにより、特に飲食料品中の細菌等の適切な加熱条件を検討することができるため、細菌の加熱殺菌理論は、産業上極めて重要であると認識されている。しかしながら、従来型の細胞の耐熱性評価法は、バイオマスとして培養した細胞を用いた耐熱性評価であるため、多種多様な細菌の混合物を用いることは困難である。そのため、従来型の細胞の耐熱性評価法では単離やその後の培養に手間を要し、生残菌数の測定も煩雑であり、細胞の耐熱性評価は大変な時間と手間を要するものであった。　 Conventionally, evaluation of heat resistance of cells has been performed by heating a sample for a certain period of time based on a general heat sterilization theory and measuring the number of surviving bacteria after heating. Thereby, since suitable heating conditions, such as bacteria in food-drinks, can be examined especially, the heat-sterilization theory of bacteria is recognized as being very important industrially. However, since the conventional method for evaluating heat resistance of cells is evaluation of heat resistance using cells cultured as biomass, it is difficult to use a mixture of a wide variety of bacteria. Therefore, the conventional method for evaluating the heat resistance of cells requires time and labor for isolation and subsequent culture, and the measurement of the number of surviving bacteria is cumbersome, and the heat resistance evaluation of cells requires a lot of time and labor. there were. *
近年、細胞の硬度を測定することにより、細胞の耐熱性を評価する方法が開発された（特許文献１）。この方法は、細胞の硬度と細胞の耐熱性とが比例することを利用した細胞の耐熱性評価法である。すなわち、各種細胞を用いて細胞の硬度と耐熱性に関して予め作成した検量線に、耐熱性未知の細胞の特定温度での硬度を当てはめることで、特定温度における当該細胞の耐熱性を評価することができる。この方法によれば、検量線さえ作成しておけば、その後は、細胞の特定温度における耐熱性を簡便かつ迅速に評価することができる。しかしながら、この方法では、評価対象が、細胞の硬度と細胞の耐熱性との比例関係が認められる芽胞形成細菌の芽胞に限られていた。例えば、好熱性偏性嫌気性芽胞形成細菌の芽胞やカビの子嚢胞子などでは、細胞の硬度と耐熱性の間に相関性が認められず、このような微生物種には適用できないという問題があった。そのため、幅広い微生物種に適用できる簡便かつ迅速な細胞の耐熱性評価法の開発が望まれていた。 In recent years, a method for evaluating the heat resistance of a cell by measuring the hardness of the cell has been developed (Patent Document 1). This method is a method for evaluating the heat resistance of cells utilizing the fact that the hardness of cells and the heat resistance of cells are proportional. That is, by applying the hardness at a specific temperature of a cell of unknown heat resistance to a calibration curve prepared in advance with respect to the hardness and heat resistance of the cell using various cells, the heat resistance of the cell at a specific temperature can be evaluated. it can. According to this method, as long as a calibration curve is prepared, the heat resistance of the cells at a specific temperature can be easily and rapidly evaluated thereafter. However, in this method, the evaluation object is limited to spores of spore-forming bacteria in which a proportional relationship between cell hardness and cell heat resistance is recognized. For example, in the case of thermophilic obligate anaerobic spore-forming bacteria spores and mold ascospores, there is no correlation between cell hardness and heat resistance, and it is not applicable to such microbial species. there were. Therefore, development of a simple and rapid method for evaluating the heat resistance of cells applicable to a wide range of microbial species has been desired.
特開２００９－１８３２０７号公報JP 2009-183207 A
本発明は、細胞の耐熱性を簡便かつ迅速に評価する方法を提供することを目的とする。 An object of this invention is to provide the method of evaluating the heat resistance of a cell simply and rapidly.
本発明者らは、細胞の形状または硬度の温度依存性を決定することによって、細胞の耐熱性を簡便かつ迅速に評価できることを見いだした。本発明は、このような知見に基づくものである。　 The present inventors have found that the heat resistance of cells can be easily and rapidly evaluated by determining the temperature dependence of the cell shape or hardness. The present invention is based on such knowledge. *
すなわち、本発明によれば、以下の発明が提供される。（１）細胞の形状または硬度の温度依存性を決定する工程を含んでなる、細胞の耐熱性評価法。（２）加熱手段を備えた走査型プローブ顕微鏡（ＳＰＭ）を用いて、細胞の形状または硬度の温度依存性を決定する、（１）に記載の方法。（３）細胞が、細菌、真菌および酵母からなる群から選択される微生物である、（１）または（２）に記載の方法。（４）所望のＤ値を示す前記細胞の加熱温度を算出する工程を更に含んでなる、（１）～（３）のいずれかに記載の方法。（５）所望の加熱温度における前記細胞のＤ値を算出する工程を更に含んでなる、（１）～（３）のいずれかに記載の方法。（６）細胞が存在する環境下における前記細胞の耐熱性を評価する、（１）～（５）のいずれかに記載の方法。　 That is, according to the present invention, the following inventions are provided. (1) A method for evaluating the heat resistance of a cell, comprising a step of determining the temperature dependence of the shape or hardness of the cell. (2) The method according to (1), wherein the temperature dependence of the cell shape or hardness is determined using a scanning probe microscope (SPM) equipped with a heating means. (3) The method according to (1) or (2), wherein the cell is a microorganism selected from the group consisting of bacteria, fungi, and yeast. (4) The method according to any one of (1) to (3), further comprising a step of calculating a heating temperature of the cell exhibiting a desired D value. (5) The method according to any one of (1) to (3), further comprising a step of calculating a D value of the cell at a desired heating temperature. (6) The method according to any one of (1) to (5), wherein the heat resistance of the cell in an environment where the cell exists is evaluated. *
本発明では、測定対象となる細胞の形状または硬度の温度依存性を決定することによって、簡便かつ迅速に細胞の耐熱性を評価することができる。細胞の単離が不要なため、細胞の混合物をそのまま測定に供することもできる。そのため、細胞の存在環境下における耐熱性を評価することができる。また、本発明によれば、任意の加熱温度におけるＤ値、または任意のＤ値を示す加熱温度を簡便かつ迅速に評価することができる。従って、本発明によれば、例えば、細菌類の存在環境下における耐熱性を評価し、飲食品の加熱処理時間を現状に即して決定することができる。 In the present invention, by determining the temperature dependence of the shape or hardness of the cell to be measured, the heat resistance of the cell can be evaluated easily and quickly. Since cell isolation is not required, the cell mixture can be used for measurement as it is. Therefore, heat resistance in the presence environment of cells can be evaluated. Moreover, according to this invention, D value in arbitrary heating temperature or the heating temperature which shows arbitrary D values can be evaluated simply and rapidly. Therefore, according to the present invention, for example, the heat resistance in the presence of bacteria can be evaluated, and the heat treatment time of the food or drink can be determined according to the current situation.
図１は、供試微生物株から調製した測定試料の細胞の加熱温度とその際の細胞の厚みの増加量との関係を示す図である。横軸は細胞の加熱温度（℃）であり、縦軸は２５℃からの細胞の厚みの増加量（ｎｍ）である。図１Ａは芽胞形成細菌の芽胞（B. licheniformisおよびB. coagulans）における結果を示し、図１Ｂは好熱性偏性嫌気性芽胞形成細菌の芽胞（T. mathraniiおよびM. thermoacetica）における結果を示し、図１Ｃは通常の細胞（栄養細胞）（E. coli、S. aureus subsp. aureusおよびS. pastrianus）における結果を示し、図１Ｄはカビの子嚢胞子（B. fulvaおよびT. flavus）における結果を示す。グラフ中の縦の線が、細胞固有の温度Ｔ１またはＴ２を求めるための直線である。FIG. 1 is a diagram showing the relationship between the cell heating temperature of the measurement sample prepared from the test microorganism strain and the increase in cell thickness at that time. The horizontal axis is the cell heating temperature (° C.), and the vertical axis is the amount of increase in cell thickness (nm) from 25 ° C. FIG. 1A shows results in spore-forming bacteria spores ( B. licheniformis and B. coagulans ), FIG. 1B shows results in thermophilic obligate anaerobic spore-forming bacteria ( T. mathranii and M. thermoacetica ), FIG. 1C shows results in normal cells (vegetative cells) ( E. coli , S. aureus subsp. Aureus and S. pastrianus ), and FIG. 1D shows results in mold ascospores ( B. fulva and T. flavus ). Indicates. The vertical line in the graph is a straight line for determining the cell-specific temperature T 1 or T 2 . 図２は、微生物の代表的な加熱致死時間曲線（ＴＤＴ曲線）を示す図である。データは、縦軸を加熱温度（℃）とし、対数目盛を有する横軸をＤ値（分）とする片対数グラフ上に示した。●は、B. licheniformis株おける加熱温度とＤ値との関係を示し、▲は、B. coagulans株における加熱温度とＤ値との関係を示す。それぞれの株に対するＴＤＴ曲線をグラフ内の直線で示す。ＴＤＴ曲線の回帰式：ｌｏｇ１０Ｄ＝ｃ＋ｄ×Ｔ　（ｃおよびｄは定数）を求めることで、Ｄ値が０．００５分となるときの加熱温度や、Ｄ値が０．０１分となるときの加熱温度を推定することが可能である（グラフ内点線）。FIG. 2 is a diagram showing a typical heat-killing time curve (TDT curve) of microorganisms. The data is shown on a semilogarithmic graph in which the vertical axis is the heating temperature (° C.) and the horizontal axis having a logarithmic scale is the D value (minutes). ● shows the relationship between the heating temperature and the D value in the B. licheniformis strain, and ▲ shows the relationship between the heating temperature and the D value in the B. coagulans strain. The TDT curve for each strain is shown as a straight line in the graph. Regression equation of TDT curve: log 10 D = c + d × T (where c and d are constants), when the D value becomes 0.005 minutes, or when the D value becomes 0.01 minutes It is possible to estimate the heating temperature (dotted line in the graph). 図３は、細胞の厚みの熱分析の際のカンチレバーの昇温速度を変化させることで、Ｔ１の温度が変化することを示す図である。毎秒１０℃または毎秒２０℃の昇温速度でB. licheniformisおよびB. coagulansにおける細胞の厚みと加熱温度との関係をそれぞれグラフに図示すると、昇温速度依存的にＴ１の温度が変化することが分かる。FIG. 3 is a diagram showing that the temperature of T 1 changes by changing the heating rate of the cantilever during the thermal analysis of the cell thickness. The relationship between cell thickness and heating temperature in B. licheniformis and B. coagulans at a rate of 10 ° C per second or 20 ° C per second is shown in the graph, and the temperature of T 1 changes depending on the rate of temperature increase. I understand.
本発明において評価対象となる細胞は、細菌などの原核生物、真菌などの真核生物、および古細菌の細胞が挙げられる。単細胞生物であればいずれの細胞でも測定対象とすることができる。また、動物や植物などの多細胞生物の細胞であっても、測定に用いることができる。単細胞生物の場合は、細胞壁の有無に関わらず測定することができる。細胞壁がある場合であっても、細胞壁の種類に関わらず測定することができ、しかも、測定に際して、細胞壁等の除去の必要はない。従って、厚い細胞壁を有するグラム陰性菌、真菌、植物、古細菌の細胞であっても、簡単な手順で、細胞の硬度または形状を測定することができる。なお、本明細書において「細胞」は、単細胞生物自体を意味するものとして用いられることがある。　 Examples of cells to be evaluated in the present invention include prokaryotes such as bacteria, eukaryotes such as fungi, and archaeal cells. Any cell can be used as a measurement target as long as it is a unicellular organism. Even cells of multicellular organisms such as animals and plants can be used for measurement. In the case of a unicellular organism, it can be measured with or without a cell wall. Even if there is a cell wall, it can be measured regardless of the type of the cell wall, and it is not necessary to remove the cell wall or the like in the measurement. Therefore, even for Gram-negative bacteria, fungi, plants, and archaea cells having a thick cell wall, the hardness or shape of the cells can be measured by a simple procedure. In the present specification, “cell” may be used to mean a unicellular organism itself. *
本発明の測定対象となる細菌としては、細菌であれば特に限定されないが、例えば、ナイセリア属（Neisseria）、ブランハメラ属（Branhamella）、ヘモフィルス属（Haemophilus）、ボルデテラ属（Bordetella）、エシェリキア属（Escherichia）、シトロバクター属（Citrobacter）、サルモネラ属（Salmonella）、シゲラ属（Shigella）、クレブシエラ属（Klebsiella）、エンテロバクター属（Enterobacter）、セラチア属（Serratia）、ハフニア属（Hafnia）、プロテウス属（Proteus）、モルガネラ属（Morganella）、プロビデンシア属（Providencia）、エルシニア属（Yersinia）、カンピロバクター属（Campylobacter）、ビブリオ属（Vibrio）、エロモナス属（Aeromonas）、シュードモナス属（Pseudomonas）、キサントモナス属（Xanthomonas）、アシネトバクター属（Acinetobacter）、フラボバクテリウム属（Flavobacterium）、ブルセラ属（Brucella）、レジオネラ属（Legionella）、ベイロネラ属（Veillonella）、バクテロイデス属（Bacteroides）およびフゾバクテリウム属（Fusobacterium）などのグラム陰性菌；ならびに、ブドウ球菌属（Staphylococcus）、レンサ球菌属（Streptococcus）、腸球菌属（Enterococcus）、コリネバクテリウム属（Corynebacterium）、バシラス属（Bacillus）、ゲオバシラス属（Geobacillus）、デスルホトマクルム属（Desulfotomaculum）、リステリア属（Listeria）、ペプトコッカス属（Peptococcus）、ペプトストレプトコッカス属（Peptostreptococcus）、クロストリジウム属（Clostridium）、ユーバクテリウム属（Eubacterium）、プロピオニバクテリウム属（Propionibacterium）、ラクトバチルス属（Lactobacillus）、ビフィドバクテリウム属（Bifidobacterium）、エンテロコッカス属（Enterococcus）、ラクトコッカス属（Lactococcus）、ペディオコッカス属（Pediococcus）、ロイコノストック属（Leuconostoc）、ストレプトコッカス属（Streptococcus）、サーモアナエロバクター属（Thermoanaerobacter）、ムーレラ属（Moorella）などのグラム陽性菌が挙げられる。測定対象としては、ブドウ球菌属、リステリア属、ラクトバチルス属、ビフィドバクテリウム属、エンテロコッカス属、ラクトコッカス属、ペディオコッカス属、ロイコノストック属、ストレプトコッカス属、ビブリオ属、サルモネラ属、エシェリキア属、カンピロバクター属、アンフィバシラス属、バシラス属、ゲオバシラス属、クロストリジウム属、デスルホトマクルム属、サーモアナエロバクター属、ムーレラ属およびスポロサルシナ属の細菌が好ましく、黄色ブドウ球菌；リステリア菌；乳酸菌；腸炎ビブリオ；サルモネラ菌；大腸菌；カンピロバクター；ならびにアンフィバシラス属、バシラス属、ゲオバシラス属、クロストリジウム属、デスルホトマクルム属、サーモアナエロバクター属、ムーレラ属およびスポロサルシナ属などの芽胞形成細菌がより好ましい。黄色ブドウ球菌Staphylococcus aureus、特にStaphylococcus aureus subsp. aureus、大腸菌Escherichia coli、およびバシラス属の芽胞形成菌であるBacillus subtilis、Bacillus subtilis ver. subtilis、Bacillus coagulans、Bacillus licheniformis、Bacillus megaterium、Geobacillus属の芽胞形成菌であるGeobacillus stearothermophillus、好熱性偏性嫌気性芽胞形成細菌であるThermoanaerobacter mathranii、Moorella thermoaceticaが最も好ましい。　 The bacterium to be measured in the present invention is not particularly limited as long as it is a bacterium. For example, the genus Neisseria, Branhamella, Haemophilus, Bordetella, Escherichia ), Citrobacter, Salmonella, Shigella, Klebsiella, Enterobacter, Serratia, Hafnia, Proteus ), Morganella, Providencia, Yersinia, Campylobacter, Vibrio, Aeromonas, Pseudomonas, Xanthomonas, Xanthomonas, Acinetobacter, Flavobacterium Gram-negative bacteria such as (Flavobacterium), Brucella, Legionella, Veillonella, Bacteroides and Fusobacterium; and Staphylococcus, Streptococcus Genus (Streptococcus), Enterococcus, Corynebacterium, Bacillus, Geobacillus, Desulfotomaculum, Listeria, Peptococcus ( Peptococcus), Peptostreptococcus, Clostridium, Eubacterium, Propionibacterium, Lactobacillus, Bifidobacterium, Enterococcus Gram positive such as (Enterococcus), Lactococcus, Pediococcus, Leuconostoc, Streptococcus, Thermoanaerobacter, Moorella Examples include bacteria. Measurement targets include Staphylococcus, Listeria, Lactobacillus, Bifidobacterium, Enterococcus, Lactococcus, Pediococcus, Leuconostoc, Streptococcus, Vibrio, Salmonella, Escherichia Bacteria of the genus Campylobacter, Amphibacillus, Bacillus, Geobacillus, Clostridium, Desulfotomacrum, Thermoanaerobacter, Murella and Sporosarcina, preferably Staphylococcus aureus; Listeria monocytogenes; Lactobacillus; Vibrio parahaemolyticus; Salmonella; Escherichia coli; Campylobacter; and Amphibacillus, Bacillus, Geobacillus, Clostridium, Desulfotomacrum, Thermoanaerobacter, Muellera and Sporosarcina Spore-forming bacterium is more preferable. Staphylococcus aureus Staphylococcus aureus, in particular Staphylococcus aureus subsp. Aureus, E. coli Escherichia coli, and a spore-forming bacterium Bacillus Bacillus subtilis, Bacillus subtilis ver. Subtilis , Bacillus coagulans, Bacillus licheniformis, Bacillus megaterium, spore-forming bacteria Geobacillus sp Most preferred are Geobacillus stearothermophillus , Thermoanaerobacter mathranii , Moorella thermoacetica , which are thermophilic obligately anaerobic spore-forming bacteria.
本発明の測定対象となる真菌としては、担子菌門（Basidiomycota）、子嚢菌門（Ascomycota）、ツボカビ門（Chytridiomycota）、ネオカリマスティクス菌門（Neocallimastigomycota）、コウマクノウキン門（Blastocladiomycota）、微胞子虫門（Microsporidia）、グロムス門（Glomeromycota）、ケカビ亜門（Mucoromycotina）、ハエカビ亜門（Entomophthoromycotina）、トリモチカビ亜門（Zoopagomycotina）、キックセラ亜門（Kickxellmycotina）などの真菌が挙げられる。測定対象としては担子菌門若しくは子嚢菌門に属する酵母、または、接合菌門、子嚢菌門若しくは担子菌門に属する真菌（カビ）が好ましい。酵母としては、例えば、サッカロマイセス属（Saccharomyces）、カンジダ属（Candida）、トルロプシス属（Torulopsis）、ザイゴサッカロマイセス属（Zygosaccharomyces）、スキゾサッカロマイセス属（Schizosaccharomyces）、ピチア属（Pichia）、ヤロウィア属（Yarrowia）、ハンセヌラ属（Hansenula）、クルイウェロマイセス属（Kluyeromyces）、デバリオマイセス属（Debaryomyces）、ゲオトリクム属（Geotrichum）、ウィッケルハミア属（Wickerhamia）、フェロマイセス属（Fellomyces）などの子嚢菌門に属する酵母および、スポロ
ボロマイセス属（Sporobolomyces）などの担子菌門に属する酵母が好ましい。サッカロマイセス属の出芽酵母であるSaccharomyces cerevisiaeおよびSaccharomyces pastrianus、ならびにスキゾサッカロマイセス属の分裂酵母であるSchizosaccharomyces pombeがより好ましい。測定対象となるカビとしては、ビソクラミス属（Byssochlamys）およびタラロセミス属（Talaromyces）などの子嚢菌門に属する真菌が好ましく、Byssochlamys fulvaおよびTalaromyces flavusがより好ましい。これらの子嚢菌門の真菌は、子嚢胞子の状態であってもそのまま本発明の測定対象となる。　 The fungi to be measured in the present invention include Basidiomycota, Ascomycota, Chytridiomycota, Neocallimastigomycota, Blastocladiomycota, microspore Examples include fungi such as Microsporidia, Glomeromycota, Mucoromycotina, Haechobi Entomophthoromycotina, Zoopagomycotina, Kickxellmycotina. As the measurement target, yeast belonging to the Basidiomycota or Ascomycota or fungus (mold) belonging to the zygomycota, Ascomycota or Basidiomycota is preferred. Examples of the yeast include, for example, the genus Saccharomyces, Candida, Torulopsis, Zygosaccharomyces, Schizosaccharomyces, Pichia, Yia, Yeasts belonging to the Ascomycota such as Hansenula, Kluyeromyces, Debaryomyces, Geotrichum, Wickerhamia and Fellomyces Yeast belonging to basidiomycetes such as Sporobolomyces is preferred. More preferred are Saccharomyces cerevisiae and Saccharomyces pastrianus which are budding yeast of the genus Saccharomyces , and Schizosaccharomyces pombe which is a fission yeast of the genus Schizosaccharomyces . The fungi to be measured are preferably fungi belonging to Ascomycota such as Byssochlamys and Talaromyces, and more preferably Byssochlamys fulva and Talaromyces flavus . These Ascomycota fungi are subject to measurement in the present invention as they are even in the state of ascospores.
本発明の測定対象となる細胞としては、単離して培養した細胞だけでなく、飲食料品中や溶液中等に混入した細胞や細胞の混合物を用いることができる。これは、本発明の測定対象となる細胞は、当該細胞の存在環境下から採取したものを直接測定に用いることができるためである。本発明では、少なくとも１つの細胞を測定できれば細胞の耐熱性の評価が可能であるため、評価したい細胞を必ずしも単離する必要はなく、当該細胞の存在する環境下での細胞の耐熱性を評価することが可能である。また、本発明の測定対象となる細胞は、当該細胞の増殖に適した培養条件下で培養してから測定に用いることもできる。各細胞の増殖に適した培養条件は、当業者であれば適宜決めることができる。細胞は、当該細胞の存在環境下から採取したものを直接測定に用いる場合も、当該細胞の増殖に適した培養条件下で培養してから測定に用いる場合も、適宜固定してから測定に用いることができる。生きた細胞を測定する固定法としては特に限定されないが、風乾固定が挙げられる。　 As the cells to be measured in the present invention, not only isolated and cultured cells, but also cells or a mixture of cells mixed in foods and drinks, solutions, and the like can be used. This is because the cells to be measured according to the present invention can be collected directly from the presence environment of the cells and used for direct measurement. In the present invention, if at least one cell can be measured, the heat resistance of the cell can be evaluated. Therefore, it is not always necessary to isolate the cell to be evaluated, and the heat resistance of the cell in the environment where the cell exists is evaluated. Is possible. In addition, the cells to be measured according to the present invention can be used for measurement after being cultured under culture conditions suitable for the growth of the cells. Those skilled in the art can appropriately determine the culture conditions suitable for the growth of each cell. Whether the cells are collected directly from the environment in which the cells exist or are used for measurement, or cultured under culture conditions suitable for the growth of the cells and then used for measurement, the cells are appropriately fixed and used for measurement. be able to. Although it does not specifically limit as a fixing method which measures a living cell, Air dry fixation is mentioned. *
本発明の細胞の耐熱性評価法では細胞の形状または硬度の温度依存性を決定することにより細胞の耐熱性を評価することができる。後記実施例に示されるように、一定応力を加えられた細胞を一定の昇温速度で加熱すると温度依存的に細胞の形状が変化し、その変化割合がある温度を境に急激に変化すること、さらには、該温度が細胞の耐熱性の指標となることが見出された。従って、本発明によれば、細胞の形状（例えば、細胞の厚みや直径（桿菌の場合は、長さおよび／または幅）のような細胞形状の変化を客観的に特定できる指標）の温度依存性を決定することにより細胞の耐熱性を評価することができる。また、本発明によれば、細胞の硬度の温度依存性を決定することにより細胞の耐熱性を評価しても良く、さらにはヤング率や弾性率のような細胞の物性値の温度依存性を決定することにより細胞の耐熱性を評価しても良い。従って、本発明によれば、細胞の形状の温度依存性、あるいは、細胞の硬度、ヤング率若しくは弾性率などの細胞の物性値の温度依存性を決定することにより細胞の耐熱性を評価することができる。以下の理論に拘束されるわけではないが、細胞を昇温条件下に置いた場合、細胞の死滅前後で細胞自体の物性が変化し、その変化を捉えることにより細胞の耐熱性を評価することが可能になると考えられる。　 In the method for evaluating the heat resistance of cells of the present invention, the heat resistance of cells can be evaluated by determining the temperature dependence of the shape or hardness of the cells. As shown in the examples below, when cells with constant stress are heated at a constant rate of temperature increase, the shape of the cells changes in a temperature-dependent manner, and the rate of change rapidly changes at a certain temperature. Furthermore, it has been found that the temperature is an indicator of the heat resistance of the cells. Therefore, according to the present invention, the temperature dependence of the cell shape (for example, an index that can objectively identify a change in cell shape such as cell thickness or diameter (length and / or width in the case of Neisseria gonorrhoeae)). By determining the sex, the heat resistance of the cells can be evaluated. In addition, according to the present invention, the heat resistance of the cell may be evaluated by determining the temperature dependency of the hardness of the cell, and further, the temperature dependency of the physical property value of the cell such as Young's modulus and elastic modulus is determined. You may evaluate the heat resistance of a cell by determining. Therefore, according to the present invention, the heat resistance of a cell is evaluated by determining the temperature dependency of the cell shape or the temperature dependency of the physical property value of the cell such as the hardness, Young's modulus or elastic modulus of the cell. Can do. Although not bound by the following theory, when cells are placed under elevated temperature, the physical properties of the cells themselves change before and after cell death, and the heat resistance of the cells is evaluated by capturing the changes. Will be possible. *
細胞の形状の温度依存性を決定する工程は、細胞に応力をかけた状態で測定してもよく、応力をかけずに測定してもよい。応力をかける場合は、特に限定されないが、応力をかけた方向の細胞の形状を測定することができる。一つの具体的態様では、細胞の形状は細胞の厚みである。　 The step of determining the temperature dependence of the cell shape may be performed in a state where stress is applied to the cell, or may be measured without applying stress. When applying stress, it is not particularly limited, but the shape of the cell in the direction in which stress is applied can be measured. In one specific embodiment, the cell shape is cell thickness. *
細胞の形状の変化を測定する装置は、細胞の形状が測定できる限り特に制限無く用いることができるが、例えば、走査型プローブ顕微鏡（ＳＰＭ）が挙げられる。また、走査型プローブ顕微鏡としては、例えば、原子間力顕微鏡（ＡＦＭ）を用いることができる。走査型プローブ顕微鏡を用いた細胞の形状の測定方法は当業者に周知である。　 An apparatus for measuring a change in cell shape can be used without particular limitation as long as the cell shape can be measured, and examples thereof include a scanning probe microscope (SPM). As the scanning probe microscope, for example, an atomic force microscope (AFM) can be used. A method for measuring the shape of a cell using a scanning probe microscope is well known to those skilled in the art. *
細胞に対する応力は、走査型プローブ顕微鏡などのカンチレバーを用いて発生させることができる。また、細胞の形状は、走査型プローブ顕微鏡などのカンチレバーを用いて測定することができる。応力の発生と細胞の形状の測定のためのカンチレバーは別々のカンチレバーとすることができるが、好ましくは、同一のカンチレバーを用いて、応力を発生させ、同時にその際の細胞の形状を測定することができる。　 The stress on the cells can be generated using a cantilever such as a scanning probe microscope. The shape of the cell can be measured using a cantilever such as a scanning probe microscope. The cantilever for stress generation and cell shape measurement can be separate cantilevers, but preferably the same cantilever is used to generate stress and simultaneously measure the cell shape at that time Can do. *
細胞の硬度は弾性率あるいはヤング率（Ｎ／ｍ２またはＰａ）により表すことができる。従って、細胞の硬度の温度依存性を決定する工程は、細胞の弾性率あるいはヤング率の温度依存性を決定する工程とすることができる。すなわち、細胞の弾性率あるいはヤング率を様々な温度で測定することで細胞の硬度の温度依存性を決定することができる。なお、弾性率とヤング率は変形のしにくさを表す物性値である点で共通することから、本明細書では両者を同義のものとして扱うこととする。　 The hardness of the cell can be represented by an elastic modulus or Young's modulus (N / m 2 or Pa). Therefore, the step of determining the temperature dependency of the cell hardness can be a step of determining the temperature dependency of the elastic modulus or Young's modulus of the cell. That is, the temperature dependence of cell hardness can be determined by measuring the elastic modulus or Young's modulus of cells at various temperatures. Note that the elastic modulus and Young's modulus are common in that they are physical property values representing the difficulty of deformation, and therefore both are treated as synonymous in this specification.
ヤング率Ｅは、一般的に、　　
Figure JPOXMLDOC01-appb-M000001
 として表すことができる。ここで、応力σは、単位面積当たりの力（Ｎ／ｍ２またはＰａ）であり、ひずみεは、応力σによる変形の単位長さ当りの伸縮量である。従って、上記数式に基づいて、細胞に負荷する応力σと細胞のひずみεから、細胞のヤング率Ｅを決定することができる。細胞のひずみεは、例えば、応力σを負荷した際の細胞の厚みを無負荷時の細胞の厚みで除して求めることができる。応力の無負荷時の細胞の厚みに温度依存性がほとんど見られないときには、細胞のひずみεに代えて、(i)応力σを負荷した際の細胞の厚み若しくは厚みの変化量、または、(ii)細胞の厚みを一定にするために要する応力σを用いて細胞のヤング率の温度依存性を決定することができる。なお、細胞のひずみεは細胞の厚みだけでなく細胞の直径（桿菌の場合は、長さおよび／または幅）などを指標にしてもよい。　 Young's modulus E is generally
Figure JPOXMLDOC01-appb-M000001
 Can be expressed as Here, the stress σ is a force per unit area (N / m 2 or Pa), and the strain ε is the amount of expansion / contraction per unit length of deformation due to the stress σ. Therefore, the Young's modulus E of the cell can be determined from the stress σ applied to the cell and the strain ε of the cell based on the above formula. The cell strain ε can be obtained, for example, by dividing the cell thickness when the stress σ is loaded by the cell thickness when no load is applied. When there is almost no temperature dependence of the cell thickness when no stress is applied, instead of the cell strain ε, (i) the amount of change in the cell thickness or thickness when stress σ is applied, or ( ii) The temperature dependence of the Young's modulus of the cell can be determined using the stress σ required for making the cell thickness constant. The cell strain ε may be determined not only by the cell thickness but also by the cell diameter (length and / or width in the case of Neisseria gonorrhoeae).
細胞の形状または硬度の温度依存性は、細胞を昇温させながらこれらを測定することにより決定することができる。細胞を昇温させる手段は特に限定されないが、例えば、細胞に接触させた試料台やカンチレバーにより昇温させることができ、あるいは加熱雰囲気下に試料を置くことでも昇温させることができる。　 The temperature dependence of cell shape or hardness can be determined by measuring these while raising the temperature of the cells. The means for raising the temperature of the cells is not particularly limited. For example, the temperature can be raised by a sample stage or cantilever brought into contact with the cells, or the temperature can be raised by placing the sample in a heated atmosphere. *
昇温は、好ましくは単位時間当りの温度変化（昇温速度）を一定にして行うことができる。昇温速度に制限はないが、例えば、毎秒０．１℃～毎秒１００℃とすることができ、毎秒１℃～毎秒５０℃とすることができ、または、毎秒１０～２０℃とすることができる。細胞の昇温と形状または硬度の測定とを独立した手段で行うこともできるが、好ましくは、加熱手段を備えたカンチレバーを用いて、昇温と形状または硬度の測定とを同時に行うことができる。加熱手段を備えたカンチレバーとしては、例えば、局所加熱型カンチレバーが挙げられ、好ましくは、局所加熱型カンチレバーを備えたＳＰＭを用いることができる。なお、細胞の形状の温度依存性を測定する際には、温度変化前後の細胞の大きさの差のように細胞形状の変化を客観的に測定できれば、細胞形状そのものを測定する必要はない。　 The temperature increase can be performed preferably with a constant temperature change (temperature increase rate) per unit time. The rate of temperature rise is not limited, but can be, for example, 0.1 ° C. per second to 100 ° C. per second, 1 ° C. per second to 50 ° C. per second, or 10 to 20 ° C. per second. it can. The temperature rise of the cell and the measurement of the shape or hardness can be performed by independent means, but preferably the temperature rise and the measurement of the shape or hardness can be performed simultaneously using a cantilever equipped with a heating means. . As a cantilever provided with a heating means, for example, a local heating type cantilever can be mentioned, and an SPM provided with a local heating type cantilever can be preferably used. When measuring the temperature dependence of the cell shape, it is not necessary to measure the cell shape itself as long as the cell shape change can be objectively measured, such as the difference in cell size before and after the temperature change. *
細胞の形状または硬度の温度依存性を決定した後に、更に細胞の形状または硬度の温度依存性が特徴的に変化する温度を決定することができる。すなわち、本発明による細胞の耐熱性の評価方法は、　ａ）細胞の形状または硬度の温度依存性を決定する工程に加えて、　ｂ）決定された形状または硬度の温度依存性から、温度依存性が特徴的に変化する温度を導出する工程をさらに含んでいてもよい。上記工程ｂ）で求められた温度依存性が特徴的に変化する温度は、その高低を比較することによって、細胞の耐熱性の比較に用いることができる。このような温度依存性が特徴的に変化する温度は以下のように導出することができる。　 After determining the temperature dependence of the cell shape or hardness, the temperature at which the temperature dependence of the cell shape or hardness characteristically changes can be determined. That is, in the method for evaluating the heat resistance of cells according to the present invention, a) In addition to the step of determining the temperature dependence of cell shape or hardness, b) the temperature dependence from the temperature dependence of the determined shape or hardness. The method may further include a step of deriving a temperature at which is characteristically changed. The temperature at which the temperature dependency obtained in step b) is characteristically changed can be used for comparison of the heat resistance of cells by comparing the levels. The temperature at which such temperature dependence changes characteristically can be derived as follows. *
細胞の形状または硬度の温度依存性が変化する温度としては、細胞の形状（例えば、厚み）、または細胞の硬度の逆数を加熱温度に対してプロットしたグラフ上で、細胞の形状、または細胞の硬度の逆数の温度に対する傾きが急激に変化する温度（本明細書中、単に「Ｔ１」（℃）ということがある）若しくは細胞の形状、または細胞の硬度の逆数が極大値をしめす温度（本明細書中、単に「Ｔ２」（℃）ということがある）が該当する。上記温度Ｔ１（℃）は、上記グラフから視覚的に求めてもよいし、視覚的に求めたＴ１の前後から任意に２点を選択し、その２点を通る直線から最も距離が遠い点の温度として求めてもよい。また、このようにして求めたＴ１値を仮のＴ１値とし、この値より高い温度域および低い温度域においてそれぞれ１本の直線の回帰式を求め、得られた２本の回帰式の交点の温度をＴ１値として求めてもよい。その他、周知の方法により折れ線回帰モデルを作成し、Ｔ１を求めてもよい。このような温度Ｔ１を有する細胞としては、例えば、芽胞形成細菌の芽胞が挙げられる。　 The temperature at which the temperature dependence of the cell shape or hardness changes is the cell shape (for example, thickness), or the cell shape or cell shape on a graph in which the reciprocal of the cell hardness is plotted against the heating temperature. The temperature at which the slope of the reciprocal hardness changes abruptly (sometimes referred to simply as “T 1 ” (° C.) in the present specification) or the shape of the cell, or the temperature at which the reciprocal of the hardness of the cell exhibits a maximum value ( In the present specification, “T 2 ” (sometimes referred to as “° C.”) is applicable. The temperature T 1 (° C.) may be obtained visually from the graph, or two points are arbitrarily selected from before and after the visually obtained T 1 , and the distance from the straight line passing through the two points is the longest. You may obtain | require as temperature of a point. Further, the T 1 value obtained in this way is set as a temporary T 1 value, and a regression equation of one straight line is obtained in each of the temperature range higher and lower than this value, and the two regression equations obtained are obtained. the intersection temperatures may be calculated as a value of T 1. Other, create a line regression model by a known method, may be obtained T 1. Examples of cells having such a temperature T 1 include spores of spore-forming bacteria.
細胞の形状、または細胞の硬度の逆数が極大値をしめす加熱温度Ｔ２（℃）は、例えば、細胞の形状、または細胞の硬度の逆数が最大値を示す温度として求めることができる。このようなＴ２を有する細胞としては、例えば、芽胞形成細菌の栄養細胞、芽胞形成細菌以外の細菌および真菌を挙げることができる。これらの細胞では、温度Ｔ２の前後でもデータが高い直線性を示すので、温度Ｔ１を決定する際と同様の方法で、２本の回帰曲線または折れ線回帰モデルから、形状、または硬度の逆数が極大値を示す加熱温度Ｔ２（℃）を求めてもよい。　 The heating temperature T 2 (° C.) at which the reciprocal of the cell shape or the cell hardness has a maximum value can be obtained, for example, as the temperature at which the reciprocal of the cell shape or the cell hardness shows the maximum value. Examples of such cells having T 2 include vegetative cells of spore-forming bacteria, bacteria other than spore-forming bacteria, and fungi. In these cells, since the data shows high linearity before and after the temperature T 2 , the shape or the reciprocal of hardness is obtained from two regression curves or a line regression model in the same manner as when the temperature T 1 is determined. The heating temperature T 2 (° C.) at which can be a maximum value may be obtained.
決定された細胞の形状または硬度の温度依存性から細胞固有の温度Ｔ１またはＴ２を導出する工程は、上記と数学的に等価な方法によっても行うことができる。数学的に等価な方法としては、測定値を逆数にして温度Ｔ１またはＴ２を導出する方法、測定値にある定数を乗じて（若しくはある定数を加算して）またはある定数で除して（若しくはある定数を減じて）あるいはこれらの加減乗除を組み合わせて温度Ｔ１またはＴ２を導出する方法などを挙げることができる。具体的には、例えば、細胞の形状、または細胞の硬度の逆数が最大値（極大値）を示す温度は、細胞の形状の逆数または硬度が最小値（極小値）を示す温度を決定することでも求めることができる。　 The step of deriving the cell-specific temperature T 1 or T 2 from the temperature dependence of the determined cell shape or hardness can also be performed by a method mathematically equivalent to the above. A mathematically equivalent method is to derive the temperature T 1 or T 2 by reversing the measured value, multiplying the measured value by a constant (or adding a certain constant), or dividing by a certain constant. A method of deriving the temperature T 1 or T 2 by combining (or subtracting a certain constant) or combining these additions, subtractions, multiplications and divisions can be mentioned. Specifically, for example, the temperature at which the reciprocal of cell shape or cell hardness shows the maximum value (maximum value) is determined as the temperature at which the reciprocal of cell shape or hardness shows the minimum value (minimum value). But you can ask.
このようにして求められたＴ１またはＴ２の温度は、その高低を比較することで、それぞれの微生物の耐熱性の評価に用いることができる。具体的には、各種微生物のＴ１またはＴ２を比較し、Ｔ１またはＴ２の温度が高いほど、耐熱性が高いと評価することができる。　 The temperature of T 1 or T 2 thus determined can be used for evaluating the heat resistance of each microorganism by comparing the temperature. Specifically, T 1 or T 2 of various microorganisms are compared, and it can be evaluated that the higher the temperature of T 1 or T 2 , the higher the heat resistance.
本発明による細胞の耐熱性の評価方法では、加熱殺菌理論において、ある温度で微生物の生残数を１０分の１とするのに必要な時間（本明細書中、単に「Ｄ値」ということがある）を求めることができ、また、あるＤ値を示す加熱温度を求めることもできる。すなわち、本発明による細胞の耐熱性の評価方法は、前述の工程ａ）および工程ｂ）に加えて、　ｃ）温度依存性が特徴的に変化する温度、すなわち、細胞固有の温度Ｔ１若しくはＴ２、が対応するＤ値を決定する工程を含んでいても良く、また、工程ｃ）に加えて、　ｄ）所望のＤ値を示す加熱温度または所望の加熱温度におけるＤ値を算出する工程を更に含んでいてもよい。さらに、工程ｄ）で求められた所望のＤ値を示す加熱温度または所望の温度におけるＤ値からは、微生物の加熱殺菌条件を決めることができる。なお、一般的な加熱殺菌理論としては、「現場必携・微生物殺菌実用データ集」第１版、３１～４２頁に記載の理論を挙げることができる。　 In the method for evaluating the heat resistance of cells according to the present invention, in the heat sterilization theory, the time required to reduce the survival number of microorganisms to one-tenth at a certain temperature (referred to simply as “D value” in the present specification). And a heating temperature showing a certain D value can also be obtained. That is, in the method for evaluating the heat resistance of a cell according to the present invention, in addition to the above steps a) and b), c) a temperature at which the temperature dependence changes characteristically, that is, a cell-specific temperature T 1 2 may include a step of determining a corresponding D value, and in addition to step c), d) a step of calculating a heating temperature indicating a desired D value or a D value at a desired heating temperature. Further, it may be included. Furthermore, the heat sterilization conditions of microorganisms can be determined from the heating temperature indicating the desired D value obtained in step d) or the D value at the desired temperature. In addition, as a general heat sterilization theory, the theory described in “First-hand collection of practical data on microorganisms / microbe sterilization”, first edition, pages 31 to 42 can be mentioned.
細胞固有の温度Ｔ１またはＴ２が対応するＤ値を決定する工程ｃ）は、以下のように行うことができる。具体的には、本発明の方法により決定したＴ１またはＴ２に対応するＤ値が如何なる値をとるかは、一般
的な微生物の加熱殺菌理論を用いることで、当業者であれば容易に求めることができる。　 The step c) of determining the D value corresponding to the cell-specific temperature T 1 or T 2 can be performed as follows. Specifically, the value of the D value corresponding to T 1 or T 2 determined by the method of the present invention can be easily determined by those skilled in the art by using a general microorganism heat sterilization theory. Can be sought.
後記実施例に示されるように、ある特定の昇温速度条件で細胞の形状または硬度の温度依存性を決定し、温度依存性が特徴的に変化する温度を決定すると、その決定された温度に対応するＤ値は菌種によらずほぼ一定の数値を取る。すなわち、昇温速度条件を一定にして工程ａ）を行い、それに引き続いて工程ｂ）を行うと、それにより決定された温度Ｔ１またはＴ２は菌種によらずある特定のＤ値に対応することになる。この知見に従えば、微生物の加熱殺菌理論に基づく一般的な方法によって、ある微生物（好ましくは２～３種の微生物）について加熱温度Ｔと微生物の生残数を１０分の１とするのに必要な時間Ｄの関係式が決定される。決定されたその関係式に、その微生物についてある特定の昇温速度条件で実施された工程ａ）および工程ｂ）で決定された温度Ｔ１またはＴ２を導入し、対応するＤ値を一旦決定すれば、その後他の微生物について決定された温度Ｔ１またはＴ２も、同じ昇温速度条件を用いて測定する限り、同様のＤ値を持つと決定できる。上記のように決定されるＤ値は、細胞の昇温速度に依存して変化することから、工程ａ）における昇温速度条件を変更するときには、昇温速度条件毎にＤ値を決定することが好ましい。　 As shown in the examples below, when the temperature dependence of the cell shape or hardness is determined under a specific heating rate condition and the temperature at which the temperature dependence changes characteristically is determined, The corresponding D value takes a substantially constant value regardless of the bacterial species. That is, when the step a) is performed with the temperature rising rate condition being constant, and then the step b) is performed, the temperature T 1 or T 2 determined thereby corresponds to a specific D value regardless of the bacterial species. Will do. According to this knowledge, the heating temperature T and the number of surviving microorganisms are reduced to 1/10 for a certain microorganism (preferably 2 to 3 kinds of microorganisms) by a general method based on the heat sterilization theory of microorganisms. The relational expression for the required time D is determined. The temperature T 1 or T 2 determined in the step a) and the step b) performed at a specific heating rate condition for the microorganism is introduced into the determined relational expression, and the corresponding D value is once determined. Then, it can be determined that the temperature T 1 or T 2 determined for other microorganisms thereafter has the same D value as long as the temperature T 1 or T 2 is measured using the same heating rate condition. Since the D value determined as described above changes depending on the temperature increase rate of the cells, when changing the temperature increase rate condition in step a), determine the D value for each temperature increase rate condition. Is preferred.
ある昇温速度条件下で決定された温度Ｔ１またはＴ２とＤ値との関係を決定するために用いる関係式は、具体的には、微生物の加熱殺菌理論に基づく一般的な方法によって微生物の様々な温度におけるＤ値を取得し、それにより得られた回帰式：ｌｏｇ１０Ｄ＝ｃ＋ｄ×Ｔ　（ｃおよびｄは定数）（この式は「加熱致死時間曲線」または「ＴＤＴ曲線」と呼ばれる）を用いることができる。ＴＤＴ曲線は測定対象の微生物が存在する環境毎に決定し、決定したＴＤＴ曲線に基づいて、温度依存性が特徴的に変化する温度、すなわち、細胞固有の温度Ｔ１若しくはＴ２に対応するＤ値を決定してもよい。この場合、その環境下（培養条件下）での温度Ｔ１若しくはＴ２に対応するＤ値と決定してもよい。　 The relational expression used to determine the relationship between the temperature T 1 or T 2 determined under a certain heating rate condition and the D value is specifically determined by a general method based on the heat sterilization theory of microorganisms. D values at various temperatures are obtained, and the resulting regression equation is: log 10 D = c + d × T (where c and d are constants) (this equation is called the “heating lethal time curve” or “TDT curve”) ) Can be used. The TDT curve is determined for each environment where the microorganism to be measured exists, and based on the determined TDT curve, the temperature at which the temperature dependence changes characteristically, that is, D corresponding to the cell-specific temperature T 1 or T 2. The value may be determined. In this case, it may be determined as D value corresponding to the temperatures T 1 or T 2 of the under the circumstances (culture conditions).
また、本発明によれば、所望のＤ値を示す加熱温度または所望の加熱温度におけるＤ値を更に算出することができる。工程ｄ）は、以下のように行うことができる。具体的には、一般的な加熱殺菌理論によれば、Ｄ値と温度Ｔは、回帰式：ｌｏｇ１０Ｄ＝ｃ＋ｄ×Ｔ　（ｃおよびｄは定数）の関係を示すことが知られている。ＴＤＴ曲線の回帰式は、以下の２つの方法により求めることができる：　（ｉ）あるＤ値を示す温度と定数ｄとからＴＤＴ曲線の回帰式を決定する方法；または、　（ｉｉ）あるＤ値を示す温度と他のＤ値を示す温度とからＴＤＴ曲線の回帰式を決定する方法。　このようにして決定された回帰式を用いることで、任意の加熱温度におけるＤ値や任意のＤ値を示す加熱温度を算出することができ、さらには、所望のＤ値を示す加熱温度や所望の加熱温度におけるＤ値を算出することができる。なお、工程ｃ）において決定された、特定の昇温速度条件に対応するＤ値が所望のＤ値である場合には、当該昇温速度条件下で細胞を測定することにより、当該所望のＤ値を示す加熱温度を算出してもよい。Ｄ値と温度Ｔとの関係は、微生物毎に変化するため、工程ｄ）を行うときは回帰式を微生物毎に決定することが好ましい。また、同一微生物であっても培養条件（存在環境）により変化する可能性があるため、この回帰式は培養条件（存在環境）毎に決定することが好ましい。　 In addition, according to the present invention, it is possible to further calculate the heating temperature showing the desired D value or the D value at the desired heating temperature. Step d) can be performed as follows. Specifically, according to the general heat sterilization theory, it is known that the D value and the temperature T show a relationship of regression equation: log 10 D = c + d × T (where c and d are constants). The regression equation of the TDT curve can be obtained by the following two methods: (i) a method of determining the regression equation of the TDT curve from a temperature indicating a certain D value and a constant d; or (ii) a certain D value. A method of determining a regression equation of a TDT curve from a temperature indicating the temperature and a temperature indicating another D value. By using the regression equation thus determined, it is possible to calculate a D value at an arbitrary heating temperature or a heating temperature indicating an arbitrary D value, and further, a heating temperature or a desired temperature indicating a desired D value. The D value at the heating temperature can be calculated. In addition, when the D value corresponding to the specific heating rate condition determined in step c) is a desired D value, the desired D value is obtained by measuring the cells under the heating rate condition. You may calculate the heating temperature which shows a value. Since the relationship between the D value and the temperature T varies for each microorganism, it is preferable to determine a regression equation for each microorganism when performing step d). Moreover, even if it is the same microorganism, since it may change with culture | cultivation conditions (existing environment), it is preferable to determine this regression formula for every culture | cultivation condition (existing environment).
上記（ｉ）の方法を用いる場合には、以下のように回帰式を決定することができる。任意の温度におけるＤ値を算出する工程において、ＴＤＴ曲線の回帰式：ｌｏｇ１０Ｄ＝ｃ＋ｄ×Ｔ　（ｃおよびｄは定数）における定数ｄを、ｄ＝－（１／Ｚ）と表すと、Ｚ値は、Ｄ値の１０倍または１０分の１の変化に対応する加熱温度の変化（℃）に相当する。既に知られた通常培養条件下におけるＤ値からＺ値（または定数ｄ）を算出することは当業者であれば可能であるから、工程ｃ）で特定のＴ１またはＴ２とそれに対応するＤ値が一つでも算出できれば、ＴＤＴ曲線の回帰式を推定することができる。そのような既に知られた通常培養条件下におけるＤ値は、たとえば、ＴｒｉＢｉｏｘ　Ｌａｂｏｒａｔｏｒｉｅｓ社が提供するＴＫＤＢデータベース（例えば、ｖｅｒ３．０）から取得することができる。細胞の培養環境が変化することで、Ｄ値およびＺ値が変動する可能性があることから、測定したい微生物の存在環境にできるだけ近い環境下で得られたデータを用いることが好ましい。また、Ｚ値を適宜補正して、微生物の存在環境下におけるＺ値を推定し、ＴＤＴ曲線の回帰式の導出に用いてもよい。　 When the above method (i) is used, the regression equation can be determined as follows. In the step of calculating the D value at an arbitrary temperature, the constant d in the regression equation of the TDT curve: log 10 D = c + d × T (where c and d are constants) is expressed as d = − (1 / Z). The value corresponds to a change in heating temperature (° C.) corresponding to a change of 10 times or 1/10 of the D value. Since it is possible for those skilled in the art to calculate the Z value (or constant d) from the already known D value under normal culture conditions, in step c) a specific T 1 or T 2 and its corresponding D If even one value can be calculated, the regression equation of the TDT curve can be estimated. The D value under such known normal culture conditions can be obtained from, for example, a TKDB database (for example, ver3.0) provided by TriBiox Laboratories. Since the D value and Z value may fluctuate due to changes in the cell culture environment, it is preferable to use data obtained in an environment as close as possible to the environment in which the microorganism to be measured exists. Alternatively, the Z value may be corrected as appropriate to estimate the Z value in the presence of microorganisms, and used to derive a regression equation for the TDT curve.
上記（ｉｉ）の方法を用いる場合は、以下のように回帰式を決定することができる。任意の温度におけるＤ値を算出する工程において、ＴＤＴ曲線の回帰式：ｌｏｇ１０Ｄ＝ｃ＋ｄ×Ｔ　（ｃおよびｄは定数）は、上記（ｉｉ）に記載されるように、あるＤ値を示す温度と他のＤ値を示す温度とから決定することができる。具体的には、異なる２以上の昇温条件で得られる細胞固有の温度Ｔ１またはＴ２とそれに対応するＤ値とからＴＤＴ曲線の回帰式を求めることができる。この際、２つ以上のＤ値のうちの１つ以上をＴＫＤＢデータベースのＤ値で代用することもできる。上記（ｉｉ）の方法は、微生物の存在環境（培養環境）下におけるＴＤＴ曲線の回帰式を正確に求められる点で有利である。　 When the above method (ii) is used, the regression equation can be determined as follows. In the step of calculating the D value at an arbitrary temperature, the regression formula of the TDT curve: log 10 D = c + d × T (where c and d are constants) indicates a certain D value as described in (ii) above. It can be determined from the temperature and the temperature showing another D value. Specifically, the regression equation of the TDT curve can be obtained from the cell-specific temperature T 1 or T 2 obtained under two or more different temperature raising conditions and the corresponding D value. At this time, one or more of the two or more D values can be substituted with the D value of the TKDB database. The method (ii) is advantageous in that a regression equation of a TDT curve can be accurately obtained in the presence environment (culture environment) of microorganisms.
本発明の方法により得られた任意の温度におけるＤ値を用いて、現実の加熱殺菌の条件を定めることができる。例えば、加熱時間を５Ｄまたは１２Ｄとすることで、生残微生物数をそれぞれ１０５または１０１２のオーダーで減少させることができる。例えば、G. stearothermophilus（B. stearothermophilus）およびC. sporogenes芽胞では、加熱殺菌時間を５Ｄとすることができる。また、命に関わる危険なボツリヌス菌芽胞では、加熱殺菌時間を１２Ｄとすることができる。また、無菌充填法で製造されるロングライフ牛乳（ＬＬ牛乳）やＰＥＴボトル清涼飲料では、加熱殺菌時間を６Ｄとすることができる。このような加熱殺菌時間は、殺菌対象となる細菌の毒性や殺菌する飲食品等の保存性を考慮して、当業者によって適宜設定することができる。 The actual heat sterilization conditions can be determined using the D value at an arbitrary temperature obtained by the method of the present invention. For example, by setting the heating time to 5D or 12D, the number of surviving microorganisms can be reduced on the order of 10 5 or 10 12 , respectively. For example, in G. stearothermophilus ( B. stearothermophilus ) and C. sporogenes spores, the heat sterilization time can be 5D. Moreover, in the dangerous botulinum spores related to life, the heat sterilization time can be set to 12D. Moreover, in the long life milk (LL milk) manufactured by an aseptic filling method or a PET bottle soft drink, the heat sterilization time can be set to 6D. Such a heat sterilization time can be appropriately set by those skilled in the art in consideration of the toxicity of bacteria to be sterilized and the preservability of food and drink to be sterilized.
以下、実施例に基づいて本発明を具体的に説明するが、本発明はこれらの例に限定されるものではない。　 EXAMPLES Hereinafter, although this invention is demonstrated concretely based on an Example, this invention is not limited to these examples. *
実施例１：加熱機能を有する走査プローブ型顕微鏡（ＳＰＭ）の構築　本願の実施例では局所加熱機能を有するＳＰＭとして、カンチレバーを局所加熱型カンチレバー（日本サーマル・コンサルティング社製）に交換したナノサーチ顕微鏡ＳＦＴ－３５００（島津製作所社製）を用いた。このカンチレバーは、試料表面に数十Ｎ～１００Ｎの任意の一定の力を加えながら、少なくとも毎秒２０℃で、２００℃または４００℃まで細胞を昇温させるために十分な昇温能力を有していた。以下の実施例では、カンチレバーにより、細胞表面の中央部に数十Ｎ～約１００Ｎの負荷をかけながら、毎秒２０℃の昇温速度で室温（２５℃）から２００℃まで（好熱性偏性嫌気性芽胞形成細菌の場合は４００℃まで）細胞を昇温させ、その際の細胞の厚みの変化（ｎｍ）を測定した。細胞の厚みまたは細胞の厚みの変化は、ＳＰＭ付属の取扱説明書などを参照して当業者に周知の方法により算出することができる。　 Example 1: Construction of a scanning probe microscope (SPM) having a heating function In this example, a nanosearch microscope in which a cantilever is replaced with a locally heated cantilever (manufactured by Nippon Thermal Consulting) as an SPM having a local heating function. SFT-3500 (manufactured by Shimadzu Corporation) was used. This cantilever has a sufficient temperature raising capability to raise the temperature to 200 ° C. or 400 ° C. at least at 20 ° C. while applying an arbitrary constant force of several tens of N to 100 N to the sample surface. It was. In the following examples, from the room temperature (25 ° C.) to 200 ° C. (thermophilic anaerobic anaerobic condition) at a rate of temperature increase of 20 ° C. per second while applying a load of several tens of N to about 100 N on the center of the cell surface by a cantilever. (In the case of sexually spore-forming bacteria, the cells were heated up to 400 ° C.), and the change (nm) in cell thickness at that time was measured. The cell thickness or the change in the cell thickness can be calculated by a method well known to those skilled in the art with reference to an instruction manual attached to the SPM.
実施例２：供試微生物株試料の調製２－１．供試微生物株　供試微生物株として、Bacillus属の芽胞形成細菌（芽胞および栄養相）、好熱性偏性嫌気性芽胞形成細菌、細菌、カビ、酵母などの幅広い生物種を用いた。具体的には、Bacillus属の芽胞形成細菌としてGeobacillus stearothermophillus　NBRC13737株、Bacillus coagulans DSM1株、Bacillus subtilis NBRC13719T株、Bacillus licheniformis NBRC12200株およびBacillus megaterium NBRC15308T株を用い、好熱性偏性嫌気性芽胞形成細菌としては、Thermoanaerobacter mathranii DSM11426株およびMoorella thermoacetica DSM521T株を用い、細菌としては、Staphylococcus aureus subsp. aureus NBRC100910株、Escherichia coli NBRC3301株およびBacillus subtilis NBRC13719T株の栄養相の細胞を用い、カビの子嚢胞子としては、Byssochlamys fulva DSM1808株およびTalaromyces flavus DSM63536株の子嚢胞子を用い、酵母としては、Saccharomyces pastorianus RIB2010株を用いた。　 Example 2: Preparation of test microorganism strain sample 2-1. Test microorganism strains As test microorganism strains, a wide variety of species such as spore-forming bacteria (spore and nutrient phase) of the genus Bacillus, thermophilic obligately anaerobic spore-forming bacteria, bacteria, fungi, and yeast were used. Specifically, Geobacillus stearothermophillus NBRC13737 strain, Bacillus coagulans DSM1 strain, Bacillus subtilis NBRC13719T strain, Bacillus licheniformis NBRC12200 strain and Bacillus megaterium NBRC15308T strain were used as the spore-forming bacterium of the genus Bacillus. , using a Thermoanaerobacter mathranii DSM11426 strain and Moorella thermoacetica DSM521T strain, the bacteria, Staphylococcus aureus subsp. aureus NBRC100910 strain, using a cell nutritional phase of Escherichia coli NBRC3301 strain and Bacillus subtilis NBRC13719T strain, as ascospores of fungi, Byssochlamys fulva DSM1808 and Talaromyces flavus DSM63536 ascospores were used, and Saccharomyces pastorianus RIB2010 was used as the yeast.
２－２．培養条件　酵母Saccharomyces pastorianus RIB2010株は、ＹＭ培地（Ｄｉｆｃｏ社製、製品番号：２７１１２０）を用いて２５℃で４８時間培養した。好熱性偏性嫌気性芽胞形成細菌は、変法ＴＧＣ培地（日水製薬社製、製品番号：３０２０５６２９３）を用いて５５℃で７２時間～９６時間培養した。その他の供試微生物株は、肉汁培地（Ｄｉｆｃｏ社製、製品番号：２３４０００）を用いて３５℃で２４時間培養した。好熱性偏性嫌気性芽胞形成細菌に関しては、嫌気ガス発生試薬アネロパック（登録商標）・嫌気培養用ガス発生剤（三菱ガス化学社製）を用いて嫌気培養を行った。　 2-2. Culture Conditions Yeast Saccharomyces pastorianus RIB2010 strain was cultured at 25 ° C. for 48 hours using YM medium (Difco, product number: 271120). Thermophilic obligate anaerobic spore-forming bacteria were cultured at 55 ° C. for 72 to 96 hours using a modified TGC medium (manufactured by Nissui Pharmaceutical Co., Ltd., product number: 30206293). Other test microorganism strains were cultured at 35 ° C. for 24 hours using a gravy medium (Difco, product number: 234000). Regarding the thermophilic obligate anaerobic spore-forming bacteria, anaerobic culture was performed using an anaerobic gas generating reagent Aneropack (registered trademark) and a gas generating agent for anaerobic culture (manufactured by Mitsubishi Gas Chemical Company).
２－３．測定用試料の調製　その後、酵母および細菌の測定用試料は、遠心分離（５，０００ｒｐｍ）により菌体を回収し、純水で２回洗浄を行うことにより、調製した。芽胞の測定用試料は、近藤雅臣、渡部一仁編「スポア実験マニュアル」技報堂出版（１９９５）の１９～３０頁に記載された方法により調製した。具体的には、５０ｍＬの肉汁培地で芽胞形成細菌を３５℃で４８時間培養して増殖させた後、遠心分離（５，０００ｒｐｍ）で回収した菌体に、１００μｇ／ｍＬのリゾチーム（和光純薬）を溶解した２００μＬの１０ｍＭトリス塩酸緩衝溶液（ｐＨ７．６）を加えた。さらに３５℃にて３０分間培養し、再び菌体を遠心分離（５，０００ｒｐｍ）で回収し、５００ｍＭ塩化ナトリウムを溶解した１ｍＬの１０ｍＭトリス塩酸緩衝溶液（ｐＨ７．６）を加えて洗浄し、遠心分離（１０，０００ｒｐｍ）で菌体を回収した。その後、さらに菌体を１０ｍＬの純水で洗浄し、遠心分離（１０，０００ｒｐｍ）で菌体を回収する工程を２回繰り返した。得られた菌体を芽胞の測定試料（芽胞試料）とした。カビの子嚢胞子の測定用試料は、高島浩介、かび検査マニュアルカラー図譜、 (株)テクノシステム（２００２）の１３０～１３１頁に記載された方法により調製した。具体的には、ポテトデキストロース培地（日水製薬社製、製品番号：３０２０５７０９２）を用いて、カビを２５℃で１ヶ月間培養し、光学顕微鏡下で子嚢胞子の形成が確認できたものをカビの子嚢胞子の測定用試料（子嚢胞子試料）とした。　 2-3. Preparation of Measurement Samples Thereafter, yeast and bacteria measurement samples were prepared by collecting cells by centrifugation (5,000 rpm) and washing twice with pure water. A sample for measuring spores was prepared by the method described on pages 19 to 30 of “Spore Experiment Manual” edited by Masaomi Kondo and Kazuhito Watanabe, published by Gihodo Publishing (1995). Specifically, after spore-forming bacteria were grown in a 50 mL broth medium for 48 hours at 35 ° C. and grown, 100 μg / mL lysozyme (Wako Pure Chemical Industries) was added to the cells recovered by centrifugation (5,000 rpm). 200 μL of 10 mM Tris-HCl buffer solution (pH 7.6) was added. The cells were further cultured at 35 ° C. for 30 minutes, and the cells were collected again by centrifugation (5,000 rpm), washed with 1 mL of 10 mM Tris-HCl buffer solution (pH 7.6) dissolved in 500 mM sodium chloride, and centrifuged. The cells were collected by separation (10,000 rpm). Then, the process which wash | cleans a microbial cell further with 10 mL pure water, and collect | recovers a microbial cell by centrifugation (10,000 rpm) was repeated twice. The obtained microbial cells were used as spore measurement samples (spore samples). A sample for measuring mold ascospores was prepared by the method described on pages 130-131 of Kosuke Takashima, mold inspection manual color chart, Techno System (2002). Specifically, using a potato dextrose medium (manufactured by Nissui Pharmaceutical Co., Ltd., product number: 302057092), mold was cultured at 25 ° C. for 1 month, and ascospore formation was confirmed under an optical microscope. A sample for measuring mold ascospores (ascospore sample) was used.
このようにして調製した細胞または芽胞懸濁液の試料をスライドグラス上に滴下し、風乾固定した。風乾固定後の試料は、直ちに測定に用いた。風乾固定後の細胞は生きた状態であると考えられる。　 A sample of the cell or spore suspension thus prepared was dropped onto a slide glass and fixed by air drying. The sample after air drying fixation was used for measurement immediately. The cells after air-drying fixation are considered to be alive. *
実施例３：熱分析による測定用試料細胞の厚みの増加量（ｎｍ）の測定　スライドグラス上で風乾固定した測定試料細胞の中心表面に、ＳＰＭの局所加熱型カンチレバーを接触させ、細胞の厚み方向に一定の力（約数十Ｎ～１００Ｎ）を加え続けた。その状態で、カンチレバーの先端温度を一定速度（毎秒２０℃）で２００℃まで（好熱性偏性嫌気性芽胞形成細菌の場合は４００℃まで）昇温させながら、細胞の厚みの増加量（ｎｍ）をモニターした。　 Example 3: Measurement of increase in thickness (nm) of sample cell for measurement by thermal analysis SPM locally heated cantilever is brought into contact with the center surface of the measurement sample cell fixed by air-drying on a slide glass, and the thickness direction of the cell A constant force (about several tens of N to 100 N) was continuously applied. In this state, while increasing the tip temperature of the cantilever to 200 ° C. at a constant rate (20 ° C. per second) (up to 400 ° C. in the case of thermophilic obligate anaerobic spore-forming bacteria), the increase in cell thickness (nm ) Was monitored.
供試微生物株における代表的な細胞の厚みの増加量（ｎｍ）と加熱温度（℃）との関係は、図１に示される
通りであった。　 The relationship between the typical increase in cell thickness (nm) in the test microorganism strain and the heating temperature (° C.) was as shown in FIG.
供試微生物株から調製した試料のうち、芽胞試料（B. licheniformisおよびB. coagulans）における細胞の厚み（ｎｍ）と加熱温度（℃）との関係を図１Ａに示す。これらの芽胞試料では、加熱温度の上昇に伴い細胞の厚みの増加量（ｎｍ）が単調増加を示す傾向にあった（図１Ａ）。いずれの芽胞試料においても、加熱温度に対する細胞の厚みの増加量（ｎｍ）のグラフの傾きが急激に低下する温度Ｔ１（℃）が存在することが分かった（図１Ａ下向き矢印）。　 FIG. 1A shows the relationship between cell thickness (nm) and heating temperature (° C.) in spore samples ( B. licheniformis and B. coagulans ) among samples prepared from the test microorganism strains. In these spore samples, the increase in cell thickness (nm) tended to show a monotonous increase with increasing heating temperature (FIG. 1A). In any spore sample, it was found that there was a temperature T 1 (° C.) at which the slope of the graph indicating the increase in cell thickness (nm) with respect to the heating temperature suddenly decreased (downward arrow in FIG. 1A).
供試微生物株から調製した試料のうち、好熱性偏性嫌気性芽胞形成細菌の芽胞の試料（T. mathraniiおよびM. thermoacetica）における細胞の厚みの増加量（ｎｍ）と加熱温度（℃）との関係を図１Ｂに示す。これらの芽胞試料では、加熱温度の上昇に伴い細胞の厚みの増加量（ｎｍ）が単調増加を示す傾向にあった（図１Ｂ）。いずれの芽胞試料においても、加熱温度に対する細胞の厚みの増加量（ｎｍ）のグラフの傾きが急激に低下する温度Ｔ１（℃）が存在することが分かった（図１Ｂ下向き矢印）。　 Among the samples prepared from the test microorganism strains , the increase in cell thickness (nm) and heating temperature (° C) in the spore samples ( T. mathranii and M. thermoacetica ) of thermophilic obligate anaerobic spore-forming bacteria The relationship is shown in FIG. 1B. In these spore samples, the increase in cell thickness (nm) tended to show a monotonous increase with increasing heating temperature (FIG. 1B). In any spore sample, it was found that there was a temperature T 1 (° C.) at which the slope of the graph indicating the increase in cell thickness (nm) with respect to the heating temperature suddenly decreased (downward arrow in FIG. 1B).
供試微生物株から調整した試料のうち、細菌の試料（E. coli、S. pastrianusおよびS. aureus subsp. aureus）における細胞の厚みの増加量（ｎｍ）と加熱温度（℃）との関係を図１Ｃに示す。図１Ｃでは、いずれの細菌試料においても加熱温度に対する細胞の厚みの増加量（ｎｍ）のグラフが山なりの形状を示す傾向が見られ、細胞の厚みの増加量（ｎｍ）が極大値（最大値）をとる温度Ｔ２（℃）が存在することが分かった（図１Ｃ下向き矢印）。　 Of the samples prepared from the test microorganism strain , the relationship between the increase in cell thickness (nm) and the heating temperature (° C) in bacterial samples ( E. coli , S. pastrianus and S. aureus subsp. Aureus ) Shown in FIG. 1C. In FIG. 1C, in any bacterial sample, there is a tendency that the graph of the increase amount (nm) of the cell thickness with respect to the heating temperature shows a mountain shape, and the increase amount (nm) of the cell thickness is the maximum value (maximum). It was found that there is a temperature T 2 (° C.) that takes (value) (downward arrow in FIG. 1C).
試供微生物株から調製した試料のうち、カビの子嚢胞子試料（T. flavusおよびB. fulva）における細胞の厚みの増加量（ｎｍ）と加熱温度（℃）との関係を図１Ｄに示す。カビの子嚢胞子試料では、加熱温度に対する細胞の厚みの増加量（ｎｍ）のグラフが山なりの形状を示す傾向が見られ、細胞の厚みの増加量（ｎｍ）が極大値（最大値）をとる温度Ｔ２（℃）が存在することが分かった（図１Ｄ下向き矢印）。　 FIG. 1D shows the relationship between the increase in cell thickness (nm) and the heating temperature (° C.) in mold ascospore samples ( T. flavus and B. fulva ) among samples prepared from the test microorganism strains. In the mold ascospore sample, the graph of the increase in cell thickness (nm) with respect to the heating temperature tends to show a mountain shape, and the increase in cell thickness (nm) is the maximum value (maximum value). It was found that there was a temperature T 2 (° C.) at which the temperature falls (FIG. 1D downward arrow).
すなわち、細胞の厚みの増加量（ｎｍ）と加熱温度（℃）との関係は、温度Ｔ１またはＴ２を境に大きく変化する。このような温度Ｔ１またはＴ２では、細胞の物性は大きく変化すると考えられる。　 That is, the relationship between the amount of increase in cell thickness (nm) and the heating temperature (° C.) varies greatly with the temperature T 1 or T 2 as a boundary. At such a temperature T 1 or T 2 , the physical properties of the cells are considered to change greatly.
実施例４：細胞の厚みの増加量（ｎｍ）と細胞の死滅との関係　温度Ｔ１またはＴ２の前後では、このような細胞の物性の変化に伴って、細胞の生理的な状態も大きく変化したものと考えられる。温度Ｔ１またはＴ２と細胞の死滅との関係を検討するために、一般的な加熱殺菌の理論（例えば、「現場必携・微生物殺菌実用データ集」第１版、３１～４２頁に記載の理論）に従って、加熱殺菌の理論におけるＤ値と温度Ｔ１またはＴ２との関係を調べた。　 Example 4 Relationship between Increase in Cell Thickness (nm) and Cell Death Before and after temperature T 1 or T 2 , the physiological state of the cell increases with the change in cell physical properties. It seems to have changed. In order to examine the relationship between the temperature T 1 or T 2 and cell death, the general theory of heat sterilization (for example, “In-Field Essential / Microbial Sterilization Practical Data Collection”, 1st edition, pages 31-42 According to the theory), the relationship between the D value in the heat sterilization theory and the temperature T 1 or T 2 was investigated.
まず、Ｄ値と加熱温度Ｔ１との関係を調べるために、一般的な手法によりB. licheniformis株およびB. coagulans株の芽胞試料におけるＤ値を測定し、加熱温度との関係をグラフ上にプロット（●：B. licheniformis株、▲：B. coagulans株）した（図２）。各微生物株におけるＴＤＴ曲線（図２の実線）の回帰式から加熱温度Ｔ１で加熱した際のＤ値の推定値を求めたところ、どちらの芽胞試料においても推定値は０．００５分であることが分かった（図２の点線）。すなわち、上記２種の芽胞試料においては、細胞の厚みの増加量（ｎｍ）の熱分析により求められたＴ１と、Ｄ値が０．００５分となる温度Ｔ（Ｄ＝０．００５）とがほぼ一致することが分かった。　 First, in order to examine the relationship between the heating temperature T 1 of the D value, the general approach to measuring the D value in the spore sample B. licheniformis strains and B. coagulans strains, the relationship between the heating temperature on the graph Plot (●: B. licheniformis strain, ▲: B. coagulans strain) (FIG. 2). When the estimated value of the D value when heated at the heating temperature T 1 was determined from the regression equation of the TDT curve (solid line in FIG. 2) in each microorganism strain, the estimated value was 0.005 minutes in both spore samples. It was found (dotted line in FIG. 2). That is, in the two types of spore samples, T 1 obtained by thermal analysis of the increase in cell thickness (nm) and a temperature T (D = 0.005) at which the D value is 0.005 minutes. Was found to be almost identical.
同様の方法により、すべての供試微生物株においてＴ１またはＴ２を測定し、ＴＤＴ曲線の回帰式から求めたＴ（Ｄ＝０．００５）と比較した。結果は表１に示される通りであった。　 By the same method, T 1 or T 2 was measured in all the test microorganism strains and compared with T (D = 0.005) obtained from the regression equation of the TDT curve. The results were as shown in Table 1.
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002
表１は、各供試微生物株における、細胞の形状の温度依存性から求めたＴ１またはＴ２の実測値（℃）と従来法で調べたＤ値およびＺ値から算出したＴ（Ｄ＝０．００５）の理論値（℃）とを比較するための表である。　 Table 1 shows T 1 or T 2 calculated from the temperature dependence of the cell shape in each test microorganism strain (° C.) and D and Z values calculated by the conventional method T (D = It is a table | surface for comparing with the theoretical value (degreeC) of 0.005).
表１で示されるように、カビ、細菌、酵母に関わらず、いずれの供試微生物株においてもＴ１およびＴ２の温度を測定することができた。また、温度Ｔ１およびＴ２は、微生物種毎に大きく異なっていた。さらに、温度Ｔ１およびＴ２とＴ（Ｄ＝０．００５）とを比較したところ、いずれの供試微生物株においても、温度Ｔ１およびＴ２とＴ（Ｄ＝０．００５）とがほぼ一致した。このように、温度Ｔ１およびＴ２とＴ（Ｄ＝０．００５）との一致は、「界」を超えた広範な微生物種（カビ（真菌）、細菌、酵母）において共通した現象であることから、細胞の厚みの増加量（ｎｍ）の熱分析は、広範な微生物種を対象とした簡便かつ迅速な耐熱性の評価の有用なツールになりうる。特に、幅広い微生物種において、細胞の厚みの増加量（ｎｍ）の熱分析によりＴ１またはＴ２を決定することで、Ｄ値が０．００５分となる温度Ｔ（Ｄ＝０．００５）を予測することが可能であった。このことから、温度Ｔ１およびＴ２の大小は、その微生物の耐熱性を評価する上で重要な判断基準となり、例えば、温度Ｔ１およびＴ２が高いほど、微生物の耐熱性が高いと評価できることが示された。今回の実施例では一例として一定応力負荷時の細胞の厚みの変化をモニターしているが、仮に、細胞の硬度、ヤング率または細胞の直径、長さ、若しくは幅、またはそれらの変化をモニターしたとしても、同様の結果が得られると考えられる。　 As shown in Table 1, the temperature of T 1 and T 2 could be measured in any test microorganism strain regardless of mold, bacteria, or yeast. Further, the temperatures T 1 and T 2 were greatly different for each microbial species. Furthermore, when comparing the temperatures T 1 and T 2 and T (D = 0.005), in any of test microorganisms strains, temperatures T 1 and T 2 and T (D = 0.005) Togahobo Matched. Thus, the agreement between temperatures T 1 and T 2 and T (D = 0.005) is a common phenomenon in a wide range of microbial species (molds, bacteria, yeasts) beyond the “world”. Therefore, thermal analysis of the increase in cell thickness (nm) can be a useful tool for simple and rapid evaluation of heat resistance for a wide range of microbial species. In particular, in a wide range of microbial species, the temperature T (D = 0.005) at which the D value becomes 0.005 minutes can be determined by determining T 1 or T 2 by thermal analysis of the increase in cell thickness (nm). It was possible to predict. Evaluation Thus, the magnitude of the temperature T 1 and T 2, an important criterion in evaluating the heat resistance of the microorganism, for example, the higher the temperature T 1 and T 2, and high heat resistance of the microorganisms It was shown that it can be done. In this example, as an example, the change in the thickness of the cell under a constant stress load is monitored. However, the hardness of the cell, the Young's modulus, or the diameter, length, or width of the cell, or a change thereof is temporarily monitored. However, it is considered that the same result can be obtained.
実施例５：熱分析において算出されるＤ値の特性　温度Ｔ１およびＴ２とＤ値との関係を更に詳細に調べるために、カンチレバーの昇温速度を変化させて、温度Ｔ１またはＴ２（℃）を決定し、Ｄ値との関係を調べた。まず、昇温速度を変化させても、温度上昇と細胞の厚みの増加量（ｎｍ）との関係から、Ｔ１またはＴ２（℃）を求められることを確認した（図３）。次に、ＴＤＴ曲線の回帰式から求められた温度Ｔ１またはＴ２に対応するＤ値を算出したところＤ値は０．０１（分）であることが分かった（図２）。　 Example 5: Characteristic temperatures T 1 and T 2 of the D value calculated in the thermal analysis and in order to investigate the relationship between the D value in more detail, the temperature rise rate of the cantilever is changed to change the temperature T 1 or T 2 (° C.) was determined, and the relationship with the D value was examined. First, it was confirmed that even if the rate of temperature increase was changed, T 1 or T 2 (° C.) could be obtained from the relationship between the temperature increase and the amount of increase in cell thickness (nm) (FIG. 3). Next, when the D value corresponding to the temperature T 1 or T 2 obtained from the regression equation of the TDT curve was calculated, it was found that the D value was 0.01 (min) (FIG. 2).
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000003
表２は、細胞の形状の温度依存性から求めたＴ１またはＴ２の実測値（℃）と従来法により算出したＴ（Ｄ＝０．００５）およびＴ（Ｄ＝０．０１）の理論値（℃）とを比較するための表である。　 Table 2 shows the measured values (° C.) of T 1 or T 2 obtained from the temperature dependence of the cell shape, and the theory of T (D = 0.005) and T (D = 0.01) calculated by the conventional method. It is a table | surface for comparing with a value (degreeC).
表２により、確かに細胞の熱分析時の昇温速度を変化させることで、異なるＤ値を示す温度を算出することが可能であることが分かった。　 From Table 2, it was found that it is possible to calculate temperatures showing different D values by changing the heating rate during thermal analysis of cells. *
微生物の加熱理論によれば、微生物は一定温度で加熱すると細胞数が１０分の１となる加熱時間Ｄ値を有し、Ｄ値は微生物の耐熱性の指標とされている。一般的に、加熱時間を短くして同等の加熱殺菌効果を得るためには、加熱温度を高める必要があり、逆に、加熱温度を低くして同等の加熱殺菌効果を得るためには、加熱時間を長くする必要がある。すなわち、同等の加熱殺菌効果を得るための加熱時間と加熱温度とは、一方を増やせば他方が減る関係にある。この点を考慮した上で、本実施例におけるこの熱分析結果を検討してみると、昇温速度を速めるほど、より短い加熱時間で温度Ｔ（ここでＴは任意の温度）に到達するので、同等の加熱殺菌効果を得るためには、結果としてより長時間の加熱が必要となり、昇温速度の速い測定環境下では、温度Ｔ１またはＴ２（℃）が上昇すると考えられる。また、一般的に、Ｔ１またはＴ２（℃）が上昇するとＤ値は低下することとなる。これが、昇温速度を速めると温度Ｔ１またはＴ２（℃）が上昇し、Ｄ値が低下する理由であると考えられる。同様に、昇温速度を遅くするほど、より長い時間をかけなければ温度Ｔに到達しないので、より低い温度で（温度Ｔに達する前に）同等の加熱殺菌効果を得ることとなり、昇温速度の遅い測定環境下では、温度Ｔ１またはＴ２（℃）が低下する。また、一般的に、Ｔ１またはＴ２（℃）が低下するとＤ値は上昇することとなる。これが、昇温速度を遅くすると温度Ｔ１またはＴ２（℃）が低下し、Ｄ値が上昇する理由であると考えられる。このように、熱分析時の昇温速度条件によって異なるＤ値を示す加熱温度を導出できるとの結果は、一般的な加熱殺菌理論によれば合理的に解釈することができる。
  According to the heating theory of microorganisms, microorganisms have a heating time D value in which the number of cells becomes 1/10 when heated at a constant temperature, and the D value is an indicator of the heat resistance of the microorganism. Generally, in order to shorten the heating time and obtain an equivalent heat sterilization effect, it is necessary to increase the heating temperature. Conversely, in order to lower the heating temperature and obtain an equivalent heat sterilization effect, You need to lengthen the time. In other words, the heating time and the heating temperature for obtaining the same heat sterilization effect have a relationship that if one is increased, the other is decreased. Considering this point, when examining the thermal analysis result in the present embodiment, the temperature T is reached in a shorter heating time (where T is an arbitrary temperature) as the heating rate is increased. In order to obtain the same heat sterilization effect, as a result, heating for a longer time is required, and the temperature T 1 or T 2 (° C.) is considered to rise in a measurement environment having a high temperature rising rate. In general, when T 1 or T 2 (° C.) increases, the D value decreases. This is considered to be the reason why the temperature T 1 or T 2 (° C.) increases and the D value decreases when the rate of temperature increase is increased. Similarly, as the rate of temperature increase is slowed down, the temperature T is not reached unless a longer time is taken. Therefore, an equivalent heat sterilization effect is obtained at a lower temperature (before reaching the temperature T). In a slow measurement environment, the temperature T 1 or T 2 (° C.) decreases. In general, when T 1 or T 2 (° C.) decreases, the D value increases. This is considered to be the reason why the temperature T 1 or T 2 (° C.) decreases and the D value increases when the rate of temperature increase is slowed down. Thus, the result that the heating temperature which shows D value which changes with temperature rising rate conditions at the time of thermal analysis can be derived | led-out can be rationally interpreted according to the general heat sterilization theory.

Claims (6)

  1. 細胞の形状または硬度の温度依存性を決定する工程を含んでなる、細胞の耐熱性評価法。 A method for evaluating the heat resistance of a cell, comprising the step of determining the temperature dependence of the shape or hardness of the cell.
  2. 加熱手段を備えた走査型プローブ型顕微鏡を用いて、細胞の形状または硬度の温度依存性を決定する、請求項１に記載の方法。 The method according to claim 1, wherein the temperature dependence of the cell shape or hardness is determined using a scanning probe microscope equipped with heating means.
  3. 細胞が、細菌、真菌および酵母からなる群から選択される微生物である、請求項１または２に記載の方法。 The method according to claim 1 or 2, wherein the cell is a microorganism selected from the group consisting of bacteria, fungi and yeast.
  4. 所望のＤ値を示す前記細胞の加熱温度を算出する工程を更に含んでなる、請求項１～３のいずれか一項に記載の方法。 The method according to any one of claims 1 to 3, further comprising a step of calculating a heating temperature of the cell exhibiting a desired D value.
  5. 所望の加熱温度における前記細胞のＤ値を算出する工程を更に含んでなる、請求項１～３のいずれか一項に記載の方法。 The method according to any one of claims 1 to 3, further comprising a step of calculating a D value of the cell at a desired heating temperature.
  6. 細胞が存在する環境下における前記細胞の耐熱性を評価する、請求項１～５のいずれか一項に記載の方法。 The method according to any one of claims 1 to 5, wherein the heat resistance of the cell in an environment in which the cell exists is evaluated.
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KOICHI NAKANISHI ET AL.: "Cho Bisho Ryoiki no Netsuoryoku ni Taisuru Creep o Riyo shita Biseibutsu Shimetsu Ondo no Chokkanteki Keisokuho no Kaihatsu - Nano-search Gijutsu no Oyo", THE CANNERS JOURNAL, vol. 90, no. 10, 1 October 2011 (2011-10-01), pages 1174 - 1175 *
KOICHI NAKANISHI ET AL.: "Gaho Saikin Tainetsusei no Jinsoku Keisokuho no Kaihatsu - Nano-search Gijutsu no Oyo Tenkai", THE CANNERS JOURNAL, vol. 89, no. 10, 1 October 2010 (2010-10-01), pages 1115 - 1116 *

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