JP4685528B2 - Pretreatment method for food samples for rapid microbial count measurement - Google Patents

Description

  The present invention relates to a food sample pretreatment method for obtaining a highly accurate measurement value when rapidly measuring the number of microorganisms in food.

Ingredient standards based on the Food Sanitation Law are stipulated for each food as “Standards for Foods, Additives, etc.”. , Food poisoning bacteria, etc.). In addition, the standard for the number of microorganisms is also defined in manufacturing standards, cooking standards, storage standards, processing standards and the like other than the component standards. Of these standards for the number of microorganisms, the total number of viable bacteria is also called the general viable count and is widely supported internationally.
The method for measuring the total number of viable bacteria is generally called the flat plate method, which is a total colony of microorganisms obtained by culturing under standard agar medium at 35 ° C. for 48 to 72 hours under aerobic (aerobic) conditions. A method of grasping with numbers is used.
However, since this method is a test for monitoring the growth of microorganisms, the time required for the inspection is long, it tends to rot such as sugar beet, and food that is shipped immediately after production can be quickly dealt with when contamination occurs. There was a drawback that it could not be taken.

  Against this background, many methods have been proposed for rapid measurement of the number of general viable bacteria, replacing the plate method. For example, an ATP method is known in which the amount of luminescence of adenosine triphosphate (ATP) in a microorganism (relative light amount: Relative Light Unit, RLU) is measured with a luminometer, and the number of microorganisms is calculated therefrom. However, since ATP is contained not only in microorganisms but also in foodstuffs, the normal ATP method has a high background value unless it is pretreated to remove ATP in foodstuffs. There was a disadvantage that it was not right.

Therefore, a method has also been proposed in which ATP in the food is removed by ATP-degrading enzyme in the ATP method, the bacterial cell membrane is dissolved to elute the ATP in the microbial cell, and the ATP luminescence reaction is carried out by firefly luciferase (non-native) Patent Document 1). However, even when the ATP erase operation is performed by this method, the background value of the ATP measurement value often shows a high value of 10 ³ RLU or more, and there are many problems in practical use.
Lucifer HS Set (Kikkoman Co., Ltd.) brochure

  The present invention has been made in view of such a conventional situation, and an object of the present invention is to quickly and easily measure the number of microorganisms in food in a fast and simple manner. It is an object to provide a method for effectively removing food from food samples.

  As a result of repeated research on a method for effectively removing a quantitative-inhibiting component derived from a food material from a food sample quickly and easily, the present inventors removed a soluble component derived from the food material or contaminants smaller than bacteria by centrifugation. The inventors have found that the above problems can be solved by removing contaminants larger than bacteria with a specific filter, and have completed the present invention.

That is, the present invention is “a food sample pretreatment method for rapid measurement of the number of microorganisms in food, wherein a liquid food sample is processed at 3900 × g to 6500 × g for 1 minute or more or 1300 × g to 5200 × After centrifuging for 3 minutes or more at 300 g or 5 minutes or more at 300 × g to 2900 × g , the precipitate suspension is passed through a track-etched membrane filter which is a filter made of a porous film having a pore diameter of 1 μm to 5 μm. The present invention provides a pretreatment method for a food sample for rapid measurement of the number of microorganisms, characterized in that the filtrate is subjected to rapid measurement as a food sample.

  By applying the present invention, it is possible to grasp the number of general viable bacteria in food quickly and with high accuracy. This greatly reduces the time required for inspection of food hygiene, and can respond to the industry's desire to ship fresh foods as quickly as possible, and reliably provide highly hygienic food to consumers. can do.

Hereinafter, the pretreatment method of the food sample for rapid microorganism count measurement of the present invention will be described based on its preferred embodiments.
In the present invention, the type of food to be measured for the number of microorganisms is not limited, but the food sample to be pretreated in the present invention needs to be liquid. For solid foods, for example, after adding sterilized distilled water to the solid food, prepare a homogenized suspension with a stomacher (registered trademark, homogenizer manufactured by Takara Sangyo Co., Ltd.) or a homogenizer. To do. The solid concentration in the suspension is preferably about 1 to 50% by mass, more preferably 5 to 20% by mass.
In a liquid food, it may be used as it is as the food sample, or may be diluted as appropriate if necessary.

  Centrifugation of the above food sample is performed at 2900 × g or more for 1 minute or more, preferably 3900 × g to 6500 × g for 1 minute or more, or 1300 × g to 5200 × g for 3 minutes or more, preferably 2000 × g It is preferable to carry out at 3900 × g for 3 minutes or more, or 300 × g-2900 × g for 5 minutes or more, preferably 700 × g-2000 × g for 5 minutes or more. The rotational speed of the rotor varies depending on the radius. For example, when a rotor having a radius of 7.2 cm is used, it is 6000 rpm or more for 1 minute or more, preferably 7000 rpm to 9000 rpm for 1 minute or more, or 4000 rpm to 8000 rpm for 3 minutes or more, preferably 5000 rpm to 7000 rpm for 3 minutes or more, Alternatively, it is 5 minutes or more at 2000 rpm to 6000 rpm, preferably 5 minutes or more at 3000 rpm to 5000 rpm.

After centrifugation, the supernatant is removed and a suspension of the resulting precipitate is prepared. It is preferable to add a washing step to the precipitate suspension again by repeating the above-mentioned “centrifugation, removal of supernatant, preparation of the resulting precipitate suspension”, and the washing step is added a plurality of times. Is more preferable.
The precipitation suspension is not particularly limited, but is preferably prepared so that the precipitation concentration does not change before and after the centrifugation operation.

The precipitate suspension thus obtained is filtered through a filter made of a porous film having a pore diameter of 1 μm to 5 μm, preferably 2 μm to 3 μm. The material of the porous film is not particularly limited, but the thickness is preferably 6 μm to 11 μm.
As a filter made of such a porous film, a commercially available track-etched membrane filter (hereinafter referred to as a TE filter) can be used. For example, an Isopore (registered trademark) TE filter manufactured by Nihon Millipore is preferably used. be able to.
As a filtration method, a pressurization method, a suction method, a centrifugal filtration method, or the like is used, but there is no particular limitation.

The filtrate obtained by the filtration is subjected to rapid measurement as a food sample. Since the filtrate contains only microorganism-derived components, by using it as a measurement sample for various rapid measurement methods, it is possible to obtain highly accurate measurement values that are almost the same as those measured by the plate culture method. .
There is no particular limitation on the rapid measurement method to which the food sample pretreated by the method of the present invention can be applied, and the ATP method and the method based on the principle of fluorescent labeling microorganisms can be suitably applied. According to the law, a luminescence reagent such as luciferase and luciferin may be added, and the ATP luminescence amount may be measured with a luminometer.

  EXAMPLES The present invention will be specifically described below with reference to examples. However, the present invention is not limited to these examples, and various embodiments are possible. The present invention is based on the concept disclosed in the present specification and drawings. It is to be understood that all embodiments are included as long as they are followed.

Preparation of test strains A Leuconostoc genus strain (hereinafter referred to as a strain) isolated from a commercially available spinach white bean and identified by a simple identification kit was prepared. Diluted as appropriate.

Examples 1-4 and Comparative Examples 1-4
Evaluation of viable cell count by pretreatment method combined with centrifugal separation and TE filter filtration Add 90ml of 0.1% peptone water to 10g of spinach white and treat with stomacher (registered trademark, homogenizer manufactured by Takara Sangyo Co., Ltd.) A 10% suspension was prepared. What added the amount of the strain shown in Table 1 to the suspension was used as a sample solution. 1 ml of each sample solution was taken in a centrifuge tube, centrifuged at 680 × g (3000 rpm) for 5 minutes (room temperature), and then the supernatant was removed. In order to wash the obtained precipitate, the precipitate was suspended in 1 ml of 0.1% peptone water, centrifuged again under the same conditions, the supernatant was removed, and the obtained precipitate was resuspended. After this washing operation was repeated once more, the washed suspension was filtered with a commercially available TE filter having a pore diameter of 3 μm (ISOPORE® TSTP filter manufactured by Nihon Millipore) (filtration method: pressurization method). The obtained filtrate was subjected to the ATP method as a food sample, and the amount of ATP emitted from the filtrate was measured with a Lumitester C-100 (manufactured by Kikkoman Corporation).
As a comparative example, 90 g of sterilized distilled water was added to 10 g of spinach white, and 10% suspension prepared by treating with a stomacher was added with the amount of the strain shown in Table 1 after centrifugation and TE filter filtration. The sample solution was directly subjected to the ATP method without performing the pretreatment by, and the ATP emission amount of the sample solution was measured with a Lumitester C-100. Moreover, the viable cell count of the sample liquid before the centrifugation process used in Examples 1-4 and the viable cell count of the sample liquid used in Comparative Examples 1-4 were measured by the plate culture method.
These measurement results are shown in Table 1. Moreover, the result of having evaluated the correlation of each viable count and luminescence amount is shown in FIG. 1 and FIG. FIG. 1 shows the results of Examples 1 to 4, and FIG. 2 shows the results of Comparative Examples 1 to 4.

  As a result, the measurement results of the ATP method using the pretreatment method combining centrifugation and TE filter filtration (results of Examples 1 to 4) are the measurement results of the ATP method without comparison (Comparative Examples 1 to 4). Compared with the number of viable bacteria measured by the plate culture method, it was confirmed that the correlation was higher.

Examples 5-8
Examination of Applicability of Sample Pretreatment Method to Other Foods For potato salad, the amount of ATP emitted and the number of viable bacteria by the plate culture method were measured in the same procedure as in Examples 1 to 4. The measurement results are shown in Table 2 and FIG.

  As a result, also in the potato salad, it was confirmed that the value of the amount of ATP luminescence by the ATP method using a pretreatment method combining centrifugation and TE filter filtration was highly correlated with the number of viable bacteria.

Examples 9-12 and Comparative Examples 5-8
Evaluation of Pretreatment in Measurement by Fluorescence A pretreated filtrate was obtained in the same manner as in Examples 1 to 4, except that the strain was added in the amount shown in Table 3. The obtained filtrate was subjected to fluorescence count using a LIVE / DEAD BacLight Bacterial Viability kit manufactured by Molecular Probes.
As a comparative example, 90 g of sterilized distilled water was added to 10 g of spinach white, and 10% suspension prepared by treating with a stomacher was added with the amount of strain shown in Table 3 by centrifugation and TE filter filtration. The sample was directly used as a sample solution for measurement of the number of bacteria by fluorescence using the above-described kit without performing the pretreatment. Moreover, the viable cell count of the sample liquid before the centrifugation process used in Examples 9-12 and the viable cell count of the sample liquid used in Comparative Examples 5-8 were measured by the plate culture method.
These measurement results are shown in Table 3.

  As a result, especially in the case of Comparative Examples 6 to 8, since the amount of fluorescence exceeded the measurement limit and measurement was impossible, food samples pretreated using the method of the present invention (Examples 9 to 12) It was confirmed that the amount of fluorescence in the filtrate was lower than that in the case of measurement without pretreatment (in the case of Comparative Examples 5 to 8), and the correlation with the viable cell count measured by the plate culture method was high. .

It is a graph which shows the result of having evaluated the correlation of the number of living bacteria and the amount of ATP luminescence in Examples 1-4. It is a graph which shows the result of having evaluated the correlation of the number of viable bacteria and the amount of ATP luminescence in Comparative Examples 1-4. It is a graph which shows the result of having evaluated the correlation of the number of living bacteria and the amount of ATP luminescence in Examples 5-8.

Claims (4)

A pretreatment method of a food sample for rapid measurement of the number of microorganisms in food, wherein a liquid food sample is 3900 × g to 6500 × g for 1 minute or more, or 1300 × g to 5200 × g for 3 minutes or more or 300 After centrifuging at xg to 2900 xg for 5 minutes or longer and passing the suspension of the precipitate through a track-etched membrane filter , which is a filter made of a porous film having a pore diameter of 1 to 5 μm, the filtrate is passed through a food sample. A method for pretreatment of a food sample for rapid measurement of the number of microorganisms, characterized by being subjected to rapid measurement as:   The pretreatment method according to claim 1, wherein the method for rapidly measuring the number of microorganisms is an ATP method.   The pretreatment method according to claim 1, wherein the rapid measurement method of the number of microorganisms is a method based on a principle of fluorescently labeling microorganisms. Pore size of the porous film is 2 m to 3 m, the pretreatment method according to any one of claims 1-3.

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