JP2812338B2 - Rapid detection of microanaerobic bacteria - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION <Industrial application field> The present invention relates to the causes of spoilage bacteria or food poisoning that cause spoilage of these products during storage, for example, in foods and beverages premised on long-term storage. The present invention relates to a method for detecting a trace anaerobic bacterium which is particularly preferably used when it is known whether or not a bacterium belonging to a so-called anaerobic bacterium such as a bacterium exists.

<Conventional technology> For example, in the case of sake that is premised on long-term storage, the presence of spoilage bacteria that can specifically grow in sake called soybean bacteria, This is a very serious problem because it will proliferate and cause the sake to spoil, and therefore, during its production, heat sterilization generally called "burning" is performed,
At the time of product shipment, it is strictly checked whether or not the bacteria are present in the product.

In addition, in the case of so-called packed foods, which have become very popular in recent years, it is premised on long-term storage,
Therefore, during its manufacture, strict sterilization treatment is applied, and at the time of packaging, special packaging such as vacuum packing is made, not only to prevent the invasion of bacteria from the outside, but also, even if trace amounts of bacteria are contained in the product. Conditions are provided such that the bacteria cannot grow even if they remain.

However, some bacteria, for example, Clostridium botulinum and C. perfringens (C)
Since there are bacteria such as lostridium perfringens that can grow even under vacuum packaging conditions, the presence of such bacteria is strictly checked during the manufacturing process.

The fire-killed bacteria, Clostridium botulinum and C. perfringens are all bacteria belonging to an anaerobic bacterium.Therefore, when detecting these bacteria, unlike the cell culture in the case of aerobic bacteria normally present in the environment, the bacterial cells Is cultured in an anaerobic atmosphere as described below, and then the number of grown colonies is visually counted.

That is, agar-containing medium spread on a Petri dish (hereinafter, referred to as
Spread a certain amount of the sample solution on an agar medium), put the chore in a closed incubator, and then adjust the carbon dioxide concentration in the incubator to 5% or more, or The pressure in the incubator is reduced to a pressure of 500 mmHg or less, and the atmosphere in the incubator is set to an anaerobic atmosphere with an oxidation-reduction potential of 0.01 V or less. This method is generally referred to as a plate agar medium method, or simply a plate method for short.

<Problems to be Solved by the Invention> However, the anaerobic bacteria as described above have a very slow growth rate as compared with the aerobic bacteria which can perform the cell culture under the atmospheric pressure, and thus are visually observed. An extremely long culture period of 3 to 15 days is required as described above until a colony grows on the medium to a size as large as possible.

In particular, in the case of those that have been sterilized at the time of manufacture, such as the above-mentioned sake and packed food, even if the above-mentioned anaerobic bacteria remain in the product, the number is several cells or less. , Or at most a very small amount of 10 cells or less, and in the case where the number of the cells themselves is very small, the growth of the cells is further deteriorated. That is the reality.

However, if it took such a long time to test for the presence of anaerobic bacteria, there was a problem with product management that the product had to be managed in a warehouse until the test was completed, or there was a problem with testing. If an anaerobic bacterium is detected as a result, problems such as the necessity of discarding all products manufactured during this time may be caused, and the economic loss is enormous. .

The present invention has been made in view of such circumstances, and at least three days or more required in the above-described conventional method,
It is an object of the present invention to provide a method for detecting trace anaerobic bacteria, which extremely shortens the examination period usually exceeding one week.

<Means for Solving the Problems> The method for detecting a trace anaerobic bacterium according to the present invention, which has been made to achieve the above object, comprises the following steps.

(A) The first step of capturing the anaerobic bacteria in the sample solution on the filter by filtering the sample solution containing the anaerobic bacteria using a filter having a pore size of 0.45 μm or less. The captured filter is placed on an agar-containing medium, and in this state, the anaerobic bacteria on the filter are cultured for 12 to 48 hours under an anaerobic atmosphere. (C) After the culture is completed, the filter is washed using a washing solution. A third step of removing the medium component attached to the filter in the second step; (d) after the washing is completed, a fourth step of measuring the amount of adenosine-3-phosphate in the anaerobic bacteria remaining on the filter. This is a detection method consisting of steps.

<Operation> Hereinafter, the present invention will be described in detail.

The method for detecting microanaerobic bacteria of the present invention comprises filtering and capturing anaerobic bacteria present in a liquid using a filter having a pore size of 0.45 μm or less, and then filtering the captured anaerobic bacteria from 12 to
By performing anaerobic cultivation in a remarkably short time as compared with the conventional method of 48 hours, the anaerobic bacteria captured on the filter can be proliferated to some extent, and then the adenosine-3 -Phosphoric acid (hereinafter AT
The basic idea is to extract and measure the amount of P, and detect anaerobic bacteria based on the measured value.

When detecting bacteria (not limited to anaerobic bacteria) present in a liquid, the bacteria are filtered and captured using a filter with a pore size of 0.45 μm or less, and then ATP in the filtered and captured bacteria is detected.
The applicant has already filed an application for a method for detecting and quantifying bacteria by directly measuring the amount (Japanese Patent Application No.
63-22075). If this method can be applied to the detection of trace anaerobic bacteria, it is possible to detect anaerobic bacteria without performing the complicated operation of culturing.

However, when the present inventor tried to apply such a direct detection method to the detection of trace anaerobic bacteria, when the number of anaerobic bacteria captured on the filter is as large as, for example, 100 or more, it is accurate. Although detection was possible, it was found that when the number was several, the amount of ATP extracted from the cells was too small to measure ATP. Of course, even when the number of anaerobic bacteria in the sample solution is small, the number of captured bacteria increases if the amount of the sample solution filtered by the filter is increased, but as a practical matter, the pore size is 0.45 μm or less. It is difficult to filter a large amount of sample liquid using a filter with an extremely fine pore diameter because of the problem of clogging of the filter, and even if it is possible, it is not applicable if there is only a small amount of sample liquid .

Therefore, the present inventors have conducted intensive research on a method that can reliably detect the bacteria even if the number of bacteria captured on the filter is as small as 10 or less, and as a result, on the filter A small amount of the captured anaerobic bacteria is anaerobically cultured in a state where the amount of ATP can be measured while being captured on the filter, and then the amount of ATP in the anaerobic bacteria that has grown is determined. Thus, the method of the present invention for measuring was completed.

The present invention will be described in detail for each step.First, in the first step, the anaerobic bacteria in the sample solution are filtered on a filter by filtering the sample solution containing the anaerobic bacteria using a filter having a pore size of 0.45 μm or less. To capture. The pore size of the filter used for filtration is usually 0.40 to 0.45 μm. However, for example, in the case of fire-killed bacteria in sake, it cannot be captured unless a pore size of 0.20 μm or less is used.

In addition, when the test target is a liquid, such as in the case of detecting bacterial fleas in sake, the liquid may be used as it is as a sample liquid. In the case of, as a pretreatment, anaerobic treatment in a solid substance is performed by using a liquid containing no bacteria, dissociated ATP and a luminescent substance (for example, a liquid containing sterilized saline or ethanol used in the third step described later). An operation of extracting the bacterium into the liquid may be performed, and the resulting extract may be used as the sample liquid.

Next, in the second step, the anaerobic bacteria captured on the filter in the first step are cultured in an anaerobic atmosphere, and are grown to an amount capable of measuring the amount of ATP in the fourth step described below.

In the anaerobic culture, the filter on which the anaerobic bacteria are captured is placed on an agar medium spread in a Petri dish or the like with the filter together with the bacteria capturing surface facing upward, and then the Petri dish is sealed in an incubator or the like. Put it inside for a predetermined time. The anaerobic conditions required for the culture may be the same as those in the conventional method. For example, carbon dioxide gas is blown into the incubator to adjust the concentration of carbon dioxide in the incubator to 5% or more, or the incubator is depressurized. The atmosphere in the incubator is adjusted to an oxidation-reduction potential of 0.01 at a pressure of -500 mmHg or less.
An anaerobic atmosphere of V or less may be used.

According to the study of the present inventors, the time required for the anaerobic culturing may be 12 to 48 hours at a culturing temperature of 25 to 60 ° C. It is wasteful from the viewpoint that bacteria do not proliferate and culture for more than 48 hours is carried out as soon as possible to detect bacteria.

Next, in the third step, the filter after completion of the culture is taken out of the incubator, and the filter is washed with a washing solution as described below.

The washing step is intended to remove the medium components attached to the filter in the second step, and more specifically, causes an error in the bacteria detection method according to the present invention, and is contained in the agar medium. The purpose is to remove free ATP from the filter and leave only the grown anaerobic bacteria on the filter. Therefore, it is necessary that the washing liquid used for the washing be a liquid that does not contain free ATP and does not contain bacteria and luminescent substances that also cause errors.
0.9% sodium chloride is added to a sterilized ethanol-containing solution obtained by dissolving ethanol to a concentration of 10% in ultrafiltration-treated pure water subjected to high-pressure steam sterilization in an autoclave at 0 ° C for 15 minutes or the ultrafiltration-treated pure water. Use a sterile saline solution (or a sterile physiological saline solution) dissolved to a concentration of.

In the fourth step, the cell membrane of the anaerobic bacteria remaining in the filter is dissolved using a lytic agent such as an enzyme or an anionic surfactant to extract ATP in the cells, and the amount of the extracted ATP Is quantified using the bioluminescence phenomenon, and anaerobic microorganisms present in the sample liquid are detected based on the ATP amount.

The method for quantifying the amount of ATP is as follows: in the presence of luciferin, which is a luminescent element, luciferase, which is a luminescent enzyme, is caused to emit light, and the amount of luminescence is electrically detected by a fluorescence detector (photoelectron detection tube); This is performed by converting it to an ATP amount.

As is clear from the above description, the method for detecting anaerobic bacteria of the present invention involves filtering and capturing anaerobic bacteria contained in a sample solution on a filter, and then culturing the captured anaerobic bacteria in an anaerobic culture. This is a method for measuring the amount of ATP in the grown anaerobic bacteria and detecting the anaerobic microorganisms present in the sample liquid. It is difficult to determine the number of anaerobic bacteria (cell number) that initially existed, and the significance of the method of the present invention is to know whether or not anaerobic bacteria were present in a sample solution within a shorter time than the conventional method. The point is that you can do it. The presence of anaerobic bacteria in foods and beverages is a serious problem itself, as described above, irrespective of its amount, and therefore, in practice, it is important to determine whether anaerobic bacteria are present. It is enough if it can be detected quickly.

<Example> An example of the present invention will be described below.

Example Sake immediately after opening (referred to as sake A), and two sakes with fungi added to each of the sakes (referred to as sake B and sake C, respectively) obtained by adding an appropriate amount of Hakufungi to the sample liquid, and 10 ml of each were used as sample liquids. The cells were filtered through a membrane filter having a pore size of μm to capture the cells.

Next, each of the above filters was placed on a petri dish in which a SI agar medium (an agar medium having an ethanol content of 5% and an agar content of 1% composition) specified by the Japan Brewing Association was spread, with the bacterial cell capturing surface facing upward. And these petri dishes were placed in a closed incubator.

After reducing the pressure in the incubator to -500 mmHg or less, further inject carbon dioxide gas in the incubator, and confirm that the inside of the incubator has an anaerobic atmosphere with an oxidation-reduction potential of 0.01 V or less (methylene blue detection After confirmation by a conventional method using a drug), the cells were cultured in this state at a temperature of 30 ° C. for 24 hours.

After the completion of the culture, each filter was taken out, and each was sufficiently washed with a sterilized ethanol-containing solution as described above, and then each filter was subjected to ATP measurement. The ATP measurement was performed using a Microsure-100 (fluorescence detector) manufactured by IMSCO. The measurement was performed three times for the same sample liquid, and the average value of the three repeated points is shown in Table 1.

As a comparative example (conventional method), 1 ml of each of the above sakes A, B, and C was taken on the same SI agar medium as described above, and the above-mentioned example was prepared according to the plate agar medium method defined by the Japan Brewing Association. Temperature 30 under the same anaerobic conditions as
After culturing at a temperature of 10 ° C., it took 10 days before the formation of colonies on the medium could be visually observed.

The number of fire-killed colonies after culturing was visually counted, and the results are shown in Table 1. Table 1 shows the average value of three repetitions of such measurement.

As is clear from Table 1, sake A in which the measured value of fire-killed bacteria in the conventional method is zero is determined by the method of the present invention.
The ATP measurement value is also zero. On the other hand, even if the measurement value of the fire-killing bacteria in the conventional method is as small as about 1 as in sake B, if the ATP is reliably detected and reduced by the method of the present invention, the fire-killing bacteria can be reduced. The presence can be reliably detected.

<Effect> According to the method of the present invention, a complicated and specialized operation such as visual counting of the number of bacterial colonies in a conventional plate method (plate agar medium method), or a long-term culture usually over one week or more is performed. All the problems that the result is not clear without it are solved, and even an anaerobic bacterium having a small amount can be detected in a significantly shorter time of 12 to 48 hours than before.

As a result, it is inevitable that it will greatly contribute to the improvement of the safety and quality of foods and beverages, etc., and will be able to manage products more efficiently than before, resulting in extremely great economic benefits. is there.

Claims (1)

(57) [Claims] 1. A method for rapid detection of trace anaerobic bacteria comprising the following steps: (A) The first step of capturing the anaerobic bacteria in the sample liquid on the filter by filtering the sample liquid containing the anaerobic bacteria using a filter having a pore size of 0.45 μm or less. (B) The anaerobic bacteria were captured. The filter is placed on an agar-containing medium, and in this state, the anaerobic bacteria on the filter are cultured under an anaerobic atmosphere for 12 to 48 hours. (C) After the culture, the filter is washed with a washing solution, Third step of removing medium components attached to the filter in the second step (d) After completion of washing, fourth step of measuring the amount of adenosine-3-phosphate in the anaerobic bacteria remaining on the filter