WO2008029645A1

WO2008029645A1 - Cell count determination method and cell count determination apparatus for thermoduric bacterium

Info

Publication number: WO2008029645A1
Application number: PCT/JP2007/066466
Authority: WO
Inventors: Chiaki Okumura; Hideki Yamanashi
Original assignee: Daikin Industries, Ltd.
Priority date: 2006-09-08
Filing date: 2007-08-24
Publication date: 2008-03-13
Also published as: TW200819533A; JP2008064648A

Abstract

Disclosed is a cell count determination method for a thermoduric bacterium, which can determine the cell count of a thermoduric bacterium within a shorter measurement time. The method comprises the following steps (A) and (B): (A) subjecting a sample to a given thermal treatment to sterilize a predetermined bacterium (a non-thermoduric bacterium such as Escherichia coli) and allow cells of a thermoduric bacterium to remain in the sample; and (B), subsequent to the step (A), adding the sample to a liquid culture medium and measuring the change in concentration of dissolved oxygen in the liquid culture medium using an oxygen electrode to determine the cell count of the thermoduric bacterium.

Description

Specification

Heat-resistant bacterial count measuring method and bacterial count measuring apparatus

Technical field

[0001] The present invention relates to a heat-resistant bacterial count measuring method and a bacterial count measuring apparatus.

Background art

For example, from the viewpoint of food hygiene management, it is required to measure the number of heat-resistant bacteria such as spore bacteria contained in food. As a method for measuring the number of heat-resistant bacteria, for example, an agar culture method has been conventionally known (Non-patent Document 1).

[0003] In the agar culture method, a predetermined heat treatment is performed, for example, at 100 ° C for 15 minutes on an agar medium to which a specimen is added. By the predetermined heat treatment, non-heat-resistant bacteria other than heat-resistant bacteria (for example, Escherichia coli, etc.) can be sterilized. Then, after the predetermined heat treatment, the heat-resistant bacteria are cultured, and the number of colonies generated is visually counted to measure the number of heat-resistant bacteria.

[0004] Non-Patent Document 1: Supervised by Toshiki Morichi, “Food Microbiology Inspection Manual”, published by Eiken Equipment Co., Ltd., July 2002, ISBN4- 9901151-1-2

Disclosure of the invention

Problems to be solved by the invention

[0005] However, the method for measuring the number of heat-resistant bacteria by the agar culture method has a problem that it takes a long time to measure. Furthermore, in the method of measuring the number of thermotolerant bacteria by the agar culture method, there are problems that the work process becomes complicated and that variations due to the work of the operator occur.

[0006] Accordingly, an object of the present invention is to provide a thermostable bacteria count measuring method and a bacteria count measuring apparatus capable of measuring the thermostable bacteria count in a shorter time / measurement time. It is another object of the present invention to provide a heat-resistant bacterial count measuring method and bacterial count measuring apparatus that are simple work and have little variation in measurement results.

Means for solving the problem

[0007] In order to achieve the above object, the thermostable bacterial count measurement according to claim 1 of the present invention The method is a thermostable count method for measuring the number of thermostable bacteria contained in a specimen, and (A) a predetermined heat treatment is performed on the specimen to sterilize the predetermined bacteria, (B) after the step (A), by adding the specimen to the liquid medium and measuring the change in the dissolved oxygen concentration of the liquid medium using an oxygen electrode, Measuring the number of heat-resistant bacteria.

[0008] The method for measuring the number of heat-resistant bacteria according to claim 2 is a method for measuring the number of heat-resistant bacteria contained in a sample, wherein (A) the liquid to which the sample is added A step of sterilizing a predetermined bacterium by subjecting the medium to a predetermined heat treatment to leave the heat-resistant bacterium in the liquid medium; and (B) after the step (A), using the oxygen electrode, the liquid And a step of measuring the number of heat-resistant bacteria by measuring a change in the dissolved oxygen concentration of the medium.

[0009] Further, the method for measuring the number of heat-resistant bacteria according to claim 3 is the method for measuring the number of heat-resistant bacteria according to claim 1 or 2, wherein the heat-resistant bacteria are spore bacteria. (C) The method further includes the step of adding pyruvic acid to the liquid medium containing the spore bacterium before the spore bacterium is grown.

[0010] Further, the bacterial count measuring apparatus according to claim 4 is a bacterial count measuring apparatus capable of measuring the heat-resistant bacterial count by the oxygen electrode method, and is a liquid installed in the bacterial count measuring apparatus. Predetermined heat treatment is applied to the liquid medium to which the oxygen electrode that can be inserted into the medium and the specimen are added, and the change in dissolved oxygen concentration in the liquid medium is measured using the oxygen electrode after the predetermined heat treatment. And a control unit for measuring the number of heat-resistant bacteria.

[0011] In the inventions according to claims 1, 2, and 4 of the present invention, the number of heat-resistant bacteria is measured by the oxygen electrode method. Therefore, the measurement time is short. In addition, it is possible to eliminate the human work required for the agar culture method, such as dilution, pour, layering, and visual count of bacteria. Therefore, the work can be simplified, and variations in measurement due to the manual work can be suppressed. Furthermore, a predetermined heat treatment is performed on the sample or the liquid medium to which the sample is added. Therefore, non-heat-resistant bacteria such as E. coli As a result, the number of bacteria can be measured only for heat-resistant bacteria.

[0012] Further, the invention according to claim 3 of the present invention further includes a step of adding pyruvic acid to a liquid medium containing the spore bacterium, before the spore bacterium is grown. Therefore

In addition, the measurement time can be further shortened and the reproducibility of the measurement results can be improved (that is, the variation in measurement can be reduced).

[0013] Objects, features, aspects, and advantages of the present invention will become more apparent from the following detailed description and the accompanying drawings.

Brief Description of Drawings

FIG. 1 is a diagram showing an example of a measurement result when the present invention is applied.

FIG. 2 is a diagram showing an example of measurement results when the present invention is applied.

FIG. 3 is a diagram when the present invention is applied to commercially available chocolate.

FIG. 4 is a measurement result diagram showing the effect of adding pyruvic acid.

BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be specifically described with reference to the drawings illustrating embodiments thereof.

Hereinafter, as an embodiment, a method for measuring the number of thermostable bacteria that can be contained in a predetermined specimen using an oxygen electrode method (DOX: dissolved oxygen electrode method) to which the present invention is applied will be described. In general, most of the heat-resistant bacteria are spore bacteria. Here, the oxygen electrode method is a method of measuring the number of microorganisms by adding a specimen to a liquid medium, culturing microorganisms, and measuring changes in the dissolved oxygen concentration of the liquid medium using an oxygen electrode. (For example, JP-A-2005-224117, JP-A-2005-304418).

[0017] Heat-resistant bacteria such as spore bacteria have strong resistance to heating, and can survive even after a predetermined heat treatment.

First, a predetermined heat treatment is performed on the specimen. By the heat treatment, predetermined bacteria (non-heat-resistant bacteria such as colon bacteria) are sterilized, and only the heat-resistant bacteria remain in the specimen. Thereafter, the specimen is added to the liquid medium. Here, the predetermined heat treatment is performed, for example, at a temperature of 100 ° C. for 15 minutes. Or apply 70 ° C for 20 minutes. As for the prescribed heat treatment, the official method is 100 ° C for 10 minutes, but for aerobic bacteria self-inspection is 70 ° C for 20 minutes, and for canned food self-inspection is 100 ° C for 15 minutes. The prescribed heat treatment is the target food, processing Depending on the method, the heating temperature and the heating time can vary (for example, 121 ° C for 20 minutes in an autoclave).

Then, the liquid medium is injected into a measurement cell having an oxygen electrode on the inner surface, and the measurement cell is set in a measurement device. The dissolved oxygen concentration in the liquid medium is converted into an electric signal by the oxygen electrode of the measuring cell, and the measuring device measures the change in the dissolved oxygen concentration in the liquid medium based on the electric signal. As a result, the measuring device can measure the initial number of heat-resistant bacteria contained in the liquid medium.

[0020] Normally, a current output from the oxygen electrode is used as the electrical signal, and the current increases as the dissolved oxygen concentration increases. Since dissolved oxygen in the liquid medium is consumed by respiration of thermotolerant bacteria, the current value decreases with time. At this time, the time required for the current value to decrease below the predetermined threshold value Ith close to zero differs depending on the initial number of bacteria, and becomes shorter as the initial number of bacteria increases. High number of heat-resistant bacteria in liquid medium

V, and so much oxygen consumption, so the dissolved oxygen concentration drops quickly. Therefore, when measuring the number of heat-resistant bacteria by the oxygen electrode method (DOX), measure the time required until the output current of the oxygen electrode reaches the threshold value Ith, and calculate the initial number of bacteria in the liquid medium from the result. I can do it.

Hereinafter, measurement results when the present invention is applied will be described.

[0022] First, the following samples were used. E. coli 100 000 (10 to the 5th power) in the diluent (saline)? A suspension of 11/1111 with 10CFU / ml, lOOOCFU / ml, and lOOOOOCFU / ml of a commercially available Bacillus subtilis spore solution is used. CFU is an abbreviation for Colony Forming Unit. For example, if 50 villages are detected from lm materials, 50 CFU / ml or 50 CFU / g is displayed.

[0023] Using the specimen, after performing the predetermined heat treatment (70 ° C for 20 minutes), the time until the number of spore bacteria is measured by the oxygen electrode method, and the predetermined heat treatment is not performed (non-heated) The time until the number of spore bacteria was measured by the oxygen electrode method was measured. The results are shown in Figs. Here, the measured values shown in Fig. 1 are plotted in Fig. 2.

[0024] As can be seen from Figs. 1 and 2, even when the present invention is applied, the number of heat-resistant bacteria (Figs. 1 and 2 Was able to measure the number of spores. When measuring the number of thermotolerant bacteria by the agar culture method, a measurement time of 48 hours is usually required. However, as shown in FIGS. 1 and 2, when the present invention is applied, any hay The number of spore bacteria could be measured in less than 24 hours at the fungal spore concentration.

In addition, as shown in FIG. 2, in the case of non-heating treatment, the measurement time does not change much even if the Bacillus subtilis spore concentration changes. From this, in the case of non-heat treatment, it can be understood that the influence involved in the measurement of non-heat-resistant bacteria such as Escherichia coli other than spore bacteria is large. On the other hand, as shown in Fig. 2, when the prescribed heat treatment is applied, the measurement time changes greatly when the concentration of Bacillus subtilis spores is changed. As a result, it can be understood that the non-thermostable bacteria such as E. coli are sterilized by the predetermined heat treatment (that is, by performing the predetermined heat treatment, it is possible to measure the number of spore bacteria alone. ).

[0026] Next, measurement of the number of heat-resistant bacteria using commercially available chocolate will be described.

[0027] A commercially available chocolate is sampled. Here, sampling in this measurement refers to the work of measuring a 5g or 10g sample from a given amount of sample and adding a 9-fold amount of physiological saline or PBS (phosphate buffer). That is. Here, physiological saline is used. After the sampling, a predetermined heat treatment at 70 ° C for 20 minutes (that is, sterilization treatment of non-heat-resistant bacteria such as E. coli) is performed and cooled.

[0028] From the cooled specimen, using a diluent (here, physiological saline), a 10-fold dilution series that is assumed to surely contain a concentration of 30-300 CFU / ml of heat-resistant bacteria make.

[0029] Thereafter, 1 ml of each of the sampling specimen (stock solution) and the 10-fold dilution series specimen is dispensed into each sterile dish. Add 15 ml of agar medium kept at 50 ° C after sterilization to each sterilized petri dish, mix well, cool and solidify, and layer 4 ml of agar medium thinly.

[0030] After 48 hours, colonies on each petri dish are visually counted, and the number of bacteria in the sample is calculated based on the dilution. The measurement result is the plate count (9.1 × 10 ³ CFU / g) in the case of “heating” in FIG.

On the other hand, the measurement result when the predetermined heat treatment is omitted among the above treatments is the plate count (6.2 × 10 ³ CFU / g) in the case of “non-heating” in FIG.

[0032] In contrast, a sample of commercially available chocolate is sampled at a predetermined temperature of 70 ° C for 20 minutes. The heat-resistant bacteria count was measured by the oxygen electrode method. The measurement result is the DOX detection time (462 ± 5 minutes) in the case of “heating” in Fig. 3. On the other hand, commercially available chocolate was sampled, and the number of heat-resistant bacteria was measured by the oxygen electrode method without performing a predetermined heat treatment. The measurement result is the DOX detection time (447 ± 27 minutes) in the case of “Non-heating” in Fig. 3.

[0033] When the DOX detection time in Fig. 3 is converted to the number of bacteria by a known method using the oxygen electrode method, the number of thermostable bacteria measured using the oxygen electrode method and the agar culture method are used. The measured number of heat-resistant bacteria (plate count) was almost the same value (when converted, non-heat-treated: 7. l X 10 ³ CFU / g, given heat treatment) : 4.5 X 10 ³ CFU / g). The relationship between the detection time and the number of bacteria is described in detail in Japanese Patent Application No. 2005-175998, Japanese Patent Application No. 11-99870, and Japanese Patent Application No. 2002-34159. Create a calibration curve by setting a threshold value (in this case, 300 nA), using the time when the current value exceeds the threshold value as the detection time, and dividing in advance, the number of bacteria or the number of bacteria measured separately. Convert the detection time to the number of bacteria.

[0034] As shown by the component force from the measurement results in Fig. 3, even when the present invention is used (that is, when thermostable bacteria are measured by the oxygen electrode method), as in the agar culture method, The number of heat-resistant bacteria present in the product can be measured. Furthermore, as can be seen from the results in FIG. 3, the measurement time can be shortened by employing the present invention.

[0035] As can be seen from the above measurement results, the number of thermostable bacteria contained in the specimen can be measured in a shorter period of time by measuring the number of thermostable bacteria using the oxygen electrode method (agar). In the case of the culture method, it is about 48 hours, but in the case of the oxygen electrode method, it is sufficiently shorter than the 48 hours). In addition, troublesome operations such as dilution, pour, layering, and visual counting of bacteria can be omitted. In addition, when the oxygen electrode method is used, the above-described dilution, pour, layering, visual counting, and other artificial work can be omitted, so that variations in measurement can be suppressed.

[0036] As can be seen from the results shown in Figs. 1 and 2, non-thermophilic bacteria (such as Escherichia coli) can be sterilized by subjecting the specimen to a predetermined heat treatment in advance. Therefore, the oxygen electrode method can be applied to a liquid medium in which only heat-resistant bacteria survive, and only the number of heat-resistant bacteria can be measured. [0037] It should be noted that the oxygen electrode method measures the number of bacteria by monitoring an electrical signal, and thus has an advantage that it is easy to automate measurement, create a database of measurement results, and network a measurement system.

[0038] Further, in the above embodiment, after non-heat-resistant bacteria are sterilized by subjecting the specimen to a predetermined heat treatment, the specimen after the predetermined heat treatment is added to the liquid medium, and the liquid medium is It was mentioned when measuring the number of heat-resistant bacteria by the oxygen electrode method.

[0039] In addition to the procedure for measuring the number of heat-resistant bacteria mentioned above, the following procedure may be adopted. Predetermined heat treatment is applied to the liquid medium to which the specimen has been added. By the predetermined heat treatment, the non-heat-resistant bacteria can be sterilized, and the heat-resistant bacteria can remain in the liquid medium. Thereafter, a change in dissolved oxygen concentration of the liquid medium subjected to the predetermined heat treatment is measured using an oxygen electrode. The number of heat-resistant bacteria can be measured by measuring the change in the dissolved oxygen concentration. According to this procedure, as described above, the number of thermotolerant bacteria contained in the specimen can be measured in a shorter time, with simple work, and while suppressing variation in measurement. Moreover, when implementing the said procedure, the following bacteria count measuring apparatuses can be provided.

That is, the bacterial count measuring apparatus can measure the number of heat-resistant bacteria by the oxygen electrode method described in, for example, the above-mentioned JP-A-2005-224117 and JP-A-2005-304418. It is a microbe count apparatus, Comprising: The following part is provided. The bacterial count measuring apparatus includes an oxygen electrode and a control unit. Here, the oxygen electrode can be inserted into a liquid medium installed in the bacterial count measuring apparatus. Further, the control unit performs control to perform a predetermined heat treatment on the liquid medium to which the specimen is added. Moreover, the said control part performs the control which measures a heat resistant microbe count by measuring the change of the dissolved oxygen concentration of a liquid culture medium using the oxygen electrode after the said predetermined heat processing.

[0041] In the bacterial count measuring apparatus, the user prepares a predetermined specimen and adds it to the liquid medium. When the liquid medium is installed in the bacterial count measuring apparatus, a predetermined heat treatment is automatically performed on the installed liquid medium under the control of the control unit. As described above, the predetermined heat treatment is, for example, 70 ° C, 20 minutes, or 100 ° C, 15 minutes, and the predetermined heat treatment can sterilize non-heat-resistant bacteria such as E. coli (in other words, In this way, only heat-resistant bacteria can remain in the liquid medium). After that, under the control of the control unit The oxygen electrode method using an oxygen electrode is automatically applied to the liquid medium, and as a result, only the number of heat-resistant bacteria can be automatically measured.

[0042] It should be noted that the method may further comprise a step of adding pyruvic acid to a liquid medium containing the spore bacterium before the growth of the spore bacterium which is a thermostable bacterium (Japanese Patent Application No. 2004-127911). ). As a result, as shown in the measurement results in FIG. 4, the measurement time can be further shortened and the reproducibility of the measurement results can be improved. Here, FIG. 4 shows the measurement results showing the DOX detection time (minutes) and the measurement variation CV (%) with and without the addition of pyruvic acid. In Figure 4, the DOX detection time is shortened and the measurement reproducibility is improved when pyruvic acid is added. In the measurement of FIG. 4, a physiological saline containing 10 ⁵ CFU / ml of coliform bacteria and 10 CFU / ml of Bacillus subtilis spore solution was used as a sample, and the sample was subjected to a predetermined heat treatment (70 ° C. for 20 minutes). did. Several samples were prepared, and some samples were added with pyruvic acid and other samples were not added with pyruvic acid.

[0043] In addition, when investigating whether heat-resistant bacteria remain in the waste! /, What! /, In order to sterilize non-heat-resistant bacteria, a predetermined heat treatment at 121 ° C for 20 minutes may be performed. . Usually, even under the prescribed heat treatment conditions, there is a possibility that even spore bacteria may be killed. However, in the center of a large volume of liquid, the temperature may not rise sufficiently, in which case the spores may survive.

[0044] In each of the above measurements, standard spore fluid was used as spore bacteria (spore fluid) (http

/ / www. eikenkizai. co. jp / pro duct / ko s oukm / ko soukm .html, Yota, http://www.wikipedia.org/wiki/% E 8% 8 A% BD% E 8% 83% 9E, Sanaki)

[0045] The present invention has been described in detail. The above description is illustrative in all aspects, and the present invention is not limited thereto. It is understood that the myriad variations S that are not illustrated can be assumed without departing from the scope of the present invention.

Claims

The scope of the claims

[1] A method for measuring the number of heat-resistant bacteria contained in a specimen,

(A) a step of sterilizing a predetermined bacterium by subjecting the specimen to a predetermined heat treatment, and leaving the thermostable bacterium in the sample;

(B) after the step (A), adding the specimen to a liquid medium, and measuring the number of heat-resistant bacteria by measuring a change in dissolved oxygen concentration of the liquid medium using an oxygen electrode; With

A method for measuring the number of heat-resistant bacteria.

[2] A method for measuring the number of heat-resistant bacteria contained in a specimen,

(A) a step of sterilizing a predetermined bacterium by applying a predetermined heat treatment to the liquid medium to which the specimen is added, and leaving the thermostable bacterium in the liquid medium;

(B) After the step (A), the step of measuring the number of heat-resistant bacteria by measuring the change in the dissolved oxygen concentration of the liquid medium using an oxygen electrode, A heat-resistant bacterial count measurement method.

[3] The heat-resistant bacterium is a spore bacterium,

(C) before the growth of the spore bacteria, further comprising the step of caloric pyruvic acid in the liquid medium containing the spore bacteria,

The method for measuring the number of heat-resistant bacteria according to claim 1 or 2, wherein:

[4] A bacterial count measuring apparatus capable of measuring the number of heat-resistant bacteria by the oxygen electrode method, wherein an oxygen electrode that can be inserted into a liquid medium installed in the bacterial count measuring apparatus and a sample are added A controller that measures the number of heat-resistant bacteria by performing a predetermined heat treatment on the liquid medium and measuring a change in the dissolved oxygen concentration of the liquid medium using the oxygen electrode after the predetermined heat treatment. With

The bacterial count measuring apparatus characterized by the above-mentioned.