JPH0121960B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for testing microorganisms on foods, water, etc.
More specifically, the present invention relates to a method for rapidly performing microbial testing using fluorescence analysis. In recent years, there has been a strong social need to prevent contamination of foods, whether fresh or processed, by microorganisms. It is legally stipulated that it must not include groups. Therefore, strict microbiological quality control is required when manufacturing and selling processed foods, and testing for these microorganisms has become extremely important. However, microbial control in the manufacturing process of processed foods and detection of microorganisms for product testing require at least 24 hours. If the time required for this detection can be reduced, it will be possible to prevent food sanitation problems, to detect and respond to hygiene control problems in food manufacturing processes early, and to improve processed food manufacturing. The benefits are extremely large. In addition, it is now clear that shortening testing time would bring great benefits not only to the manufacturing of processed foods but also to fields that require microbial testing, such as clinical microbial testing, pharmaceutical manufacturing, and water quality control in many manufacturing industries. Needless to say. Conventional bacterial count testing methods include the agar plate smear method, the agar plate pour method, and the liquid medium serial dilution method (most probable method). In all of these conventional methods, it is necessary to dilute the specimen at a certain ratio, inoculate it into a nutrient medium, and grow it until the growth of microorganisms can be determined with the naked eye.
A minimum of 24 hours is required to measure the number of bacteria.
Incidentally, the testing methods for coliform bacteria stipulated by the Food Sanitation Act differ depending on the food, but include lactose broth or brilliant green lactose broth (BGLB),
It is stipulated that culture should be carried out for 24 hours (plus/minus 2 hours) in desoxycholic acid medium or Endo medium, etc., and if it is not determined to be coliform positive, culture should be continued for an additional 48 hours (plus/minus 3 hours) before making a determination. ing. In addition, the Water Pollution Control Law stipulates that the number of coliform bacteria in wastewater is determined from the number of typical colonies that appear after culturing for 18 to 20 hours using an agar medium containing sodium desoxycholate. Furthermore, as a method for measuring the number of large intestine bacterial groups in order to maintain environmental standards related to water pollution, it is stipulated by the Environment Agency Notification No. 59 of December 28, 1971 that the most accurate method be used for measurement. There is. Recently, methods for rapidly detecting the number of microorganisms include measuring changes in the impedance of the culture medium due to the growth of microorganisms, changes in the pH of the culture solution, and the amount of oxygen consumed or carbon dioxide gas generated, and correlating these with the number of microorganisms. Research has been conducted on methods to determine the number of microorganisms from microorganisms, but in either case, detection is not possible unless the number of microorganisms is ¹⁰⁵ or more per ml of culture medium, and the above amount varies depending on the non-living substances derived from the sample. This method cannot be said to be satisfactory in terms of the detection limit and detection accuracy of microorganisms, and has not been put into practical use. Another method is to directly observe the specimen under a microscope to determine the number of microorganisms, but in this case, it is not possible to distinguish whether the microorganisms are alive or dead, and it is difficult to distinguish between non-microbial contaminants and microorganisms derived from the specimen. The method of detecting microorganisms by direct microscopic observation is also not a satisfactory method. In view of these circumstances, the present inventors have conducted various studies with the aim of developing a method for detecting the number of viable microorganisms in a specimen in an extremely short period of time.
So-called common bacteria such as yeast or filamentous fungi hydrolyze non-fluorescent umbelliferone derivatives to release fluorescent umbelliferone or its derivatives, and the intensity of fluorescence of the reaction solution at this time, that is, the hydrolysis activity of umbelliferone derivatives, The inventors discovered that the microorganisms are well proportional to the number of bacteria, and based on this relationship, trace amounts of microorganisms contained in a sample can be detected extremely quickly, leading to the completion of the present invention. That is, the gist of the present invention is to add a non-fluorescent umbelliferone derivative to a test sample containing a certain amount of specimen, cause the reaction to occur, measure the fluorescence intensity of the reaction solution, and measure the number of microorganisms based on the fluorescence intensity. It is a rapid microbial testing method. The general viable bacteria as used in the present invention mainly refers to living aerobic bacteria and fungi that exist in foods, water, medicines, etc., such as Pseudomonas, Flavobacterium, Achromobacter, Alcaligenes, and Acetobacter. genus, genus Esierisia;
Gram-negative bacilli belonging to the genus Proteus, Salmonella, Serratia, Erwinia, etc., Arthrobacter, Brevibacterium, Corynebacterium, Streptococcus, Micrococcus, Bacillus, Staphylococcus, Gram-positive bacteria belonging to the genus Lactobacillus, yeasts belonging to the genus Satucharomyces, Candeida, Torula, Pichia, Hansenula, etc., or filamentous fungi such as Aspergillus, Penicillium, Nocardia, etc., on a standard agar medium. A group of microorganisms that grow. Coliform bacteria as used in the present invention refers to a group of Gram-negative, nonspore-forming bacilli that are aerobic or facultatively anaerobic and that decompose lactose to generate gas. , a group of bacteria belonging to the genus Proteus, or Salmonella, etc. The non-fluorescent umbelliferone derivatives used in the present invention are derivatives of umbelliferone (7-hydroxycoumarin) or 4-methylumbelliferone, such as 4-methylumbelliferon phosphate (MUP), 4-methylumbelliferon. -α-D-glucopyranoside (MUGul), 4
-Methylumbelliferylacetamide-β-D
-Glucopyranoside (MUAG), 4-methylumbelliferyl-β-D-galactoside (MUβGal), 4-methylumbelliferyl-α
-D-galactoside (MUαGal), 4-methylumbelliferyl arabinoside (MUAla),
Examples include 4-methylumbelliferyl pyrophosphate (MUPP) and 4-methylumbelliferyl acetate (MUAce). Items subjected to microbial testing include fresh foods, processed foods, frozen foods, wastewater, drinking water, river water, soft drinks,
All kinds of products, including dairy products, blood, urine, biological tissues, pharmaceuticals, and cosmetics, can be tested, regardless of whether they are solids or liquids. The processing method for testing may be carried out in accordance with the procedures established in conventional microbial testing methods. When the specimen to be subjected to microbial testing is a solid substance such as food, it should be homogenized by adding sterile water, etc., and then diluted appropriately if necessary.If the specimen is a solution such as water or wastewater, it can be used as is. Add 10 ^-3 to ^{10 -4} M of the substrate to this test solution for 20 to 20 minutes.
The reaction is carried out at 50°C and pH 4 to 8 for about 30 minutes to 6 hours.
When 10 ² to ^{10 4} microorganisms/ml survive in the reaction solution, the aforementioned substrate is hydrolyzed and fluorescent umbelliferone derivatives, such as 4-methylumbelliferone (4-MU), are liberated. Therefore, the intensity of this fluorescence is measured according to a conventional method. Since the degree of fluorescence is well proportional to the number of microorganisms in the reaction solution, if the relationship between the degree of fluorescence and the number of bacteria is determined in advance, the number of bacteria in the sample can be quickly detected using this relationship. When 4-MU is irradiated with ultraviolet light having a wavelength of 360 nm, it is excited and emits fluorescence of 450 nm, and if this fluorescence is 10 ^-8 M, it can be measured by fluorescence analysis. In addition, when culturing the specimen in a culture medium, it is desirable to add a fluorescent umbelliferon derivative to the medium in advance and perform the culture, which can shorten the measurement time. Since the number of microorganisms that can liberate 10 ^-8 4-MU is 10 ³ to 10 ⁴ microorganisms/ml in the reaction solution, according to the method of the present invention, microbial testing can be performed in 1 to 2 times.
It is possible to do it in hours. Experimental Example A specific strain of common bacteria was selected and its hydrolysis activity towards non-fluorescent umbelliferone derivatives was investigated. 10 ^-3 M of 4-methylumbelliferone derivative was added to 0.5% peptone water (PH7.0), dispensed into 3.0 ml test tubes (20 ml volume), and filtered through a Millipore filter. This was inoculated with 10 to 100 cells of the following microorganisms, and cultured with shaking at 30°C for bacteria and 25°C for filamentous fungi and yeast for 24 hours. The number of bacteria in the culture solution varies depending on the strain, but it is 10 ^-6 per ml.
There were 10 ^-9 pieces.

[Table] The pH was adjusted to 11.0 by adding alkali to the culture solution obtained in this way, and then centrifuged to remove insoluble substances, excited by irradiation with 360 nm ultraviolet rays, and fluorescence at 450 nm. was measured using a fluorometer to determine the amount of 4-MU produced. The result is the first
Shown in the table.

[Table] As shown in Table 1, MUP is hydrolyzed by all bacterial strains and has strong activity, followed by MUPP,
MUAG, MUGul, MUAce, MUGal, MUAla,
A considerable amount of water was hydrolyzed. Therefore, these substrates are used for the purpose of measuring general bacteria, and MUP is a particularly desirable substrate. In order to further increase sensitivity and reliability, it is desirable to use a mixture of two or more of these, and the measurement time can also be shortened. On the other hand, MUGal,
Since MUAla, etc. are specifically hydrolyzed by coliform bacteria, they can be used to selectively measure coliform bacteria. Some enzymes that catalyze the hydrolysis of substrates exist in bacterial cells, so in such cases it is desirable to extract the intracellular enzymes using conventional methods. Sensitivity can be increased by applying Furthermore, in order to accurately measure the number of small numbers of bacteria, there are methods such as concentrating the microbial cells in the sample and prolonging the reaction time. However, although these methods are effective to a certain extent, for more accurate measurements it is better to test according to the most probable method or the culture method. Conventionally, in the most reliable method for measuring coliform bacteria, the test solution was serially diluted to 1/10 in four steps, and five bottles of each sample were transferred to a brilliant green lactose broth (BGLB) fermentation tube medium at 35-37℃. After incubation for 48 hours (±3 hours), check how many gases were generated for each diluted sample, and use the most probable number table to calculate the number of samples for 100.
It is a method of measuring the coliform bacteria in a ml, and is said to be a method that more accurately measures the number of bacteria in decimal numbers compared to methods such as the agar subtraction method. In contrast, in the method of the present invention, each diluted sample water is cultured for 2 to 12 hours in 5 bottles of a medium containing MU derivatives, and the culture supernatant is examined for the presence or absence of fluorescence. Based on the number, test using the most probable number table. Depending on the specimen, it is also possible to add a 4-MU derivative to the culture solution, react for 1 to 2 hours, and measure the fluorescence of this reaction solution. When testing the number of viable bacteria, the nutrient medium used at this time is the usual culture medium used for general viable bacteria measurements, such as peptone water, heart infusion broth, nutrient broth, etc. In order to selectively detect filamentous fungi, a medium suitable for the growth of filamentous fungi, for example, a pineapple culture medium, a malt medium, a potato dextrose medium, a cornmeal medium, etc., is used. In addition, in order to selectively culture yeast and filamentous fungi, a drug that suppresses the growth of bacteria, such as chloranephenicol or penicillin, may be added to the medium. When it is desired to detect only specific microorganisms such as coliform bacteria in a sample, sodium desoxycholate or liver juice acid may be added to the nutrient medium to suppress the growth of microorganisms other than coliform bacteria. In addition, it is possible to selectively target bacteria to be measured by adding drugs such as various antibiotics or azides to the culture medium and making use of the differences in sensitivity or resistance of microorganisms to these, or by combining these methods as appropriate. It is also possible to measure When measuring coliform bacteria using 4-methylumbelliferyl galactoside as a substrate, the galactosidase inducers lactose-isopropyl-β-D-thiogalactopyranoside (IPTG), propyl- It is desirable to add β-D-thiogalactopyranoside (PTG) or MUGal. It is preferable to culture under aerobic conditions, and the culture temperature should be within the temperature range suitable for the growth of normal bacteria (10
~40℃). When targeting a specific microorganism, it is sufficient to select culture conditions suitable for that specific microorganism.For example, when trying to selectively detect only Escherichia coli, use the difference in growth temperature to increase the temperature of 44.5±0.2 It is desirable to culture at ℃. Even with methods other than the optimal method, as shown in Example 4, if the correlation between the number of bacteria and the amount of 4-MU produced in the culture solution after culturing the specimen in a nutrient medium for a certain period of time is determined, the bacteria in the specimen can be It is also possible to measure the number directly. As described above, the present invention is a microorganism detection method using fluorescence analysis to measure ultra-trace amounts of enzyme activity, and can be widely used in the fields of food, cosmetics, medicine, and environmental conservation. Examples will be described below. Example 1 10ml of river water, well water, food factory wastewater, city sewage
Four samples were taken from each destination, and bacteria were collected by centrifugation. 5.0 ml of a phosphate buffer with a pH of 7.0 containing 10 ^-3 M of MUP was added thereto and maintained at 37°C for 2.0 hours. Next, the reaction solution was centrifuged to remove insoluble matter, the pH of the supernatant was adjusted to 11.0, and the amount of 4-MU produced in the reaction solution was determined using the same method as shown in the experimental example. Ta. On the other hand, the number of common bacteria contained in each test water was determined using a standard agar medium according to a conventional method. The relationship between the two is shown in Table 2.

【table】

[Table] As shown in Table 2, it is recognized that there is a correlation between the number of viable bacteria and the value of 4-MU. By using this relationship, the number of viable bacteria in various test water can be easily tested. Example 2 Weigh 10g of commercially available potato salad and add 90ml of sterile water.
Add and homogenize (20000rpm.1 minute),
This was used as a specimen. Take 0.1ml, 0.01ml, and 0.001ml of this, add each to 0.5% peptone water (PH7.0, 9.0ml) containing three bottles of 4-MUP at 10 ^-3 M, and shake at 30°C for 12 hours. Cultured. Add alkali to culture solution to raise pH to 10.5
After adjusting the concentration, insoluble substances were removed by centrifugation, the presence or absence of fluorescence in the supernatant was determined, and the number of bacteria was determined using the most probable number table. The same specimen was grown in standard medium (yeast extract 0.25%, peptone 0.5%, glucose
0.1%, pH 7.1) for 48 hours, the presence or absence of growth was determined by turbidity, and the number of bacteria was determined using the most probable number table. Commercially available macaroni salad and spaghetti salad were similarly tested, and the results are shown in Table 3.

[Table] For the above potato salad, instead of MUP
MUP10 ^-3 M, MUGul10 ^-3 M, and
Measurement was performed using the same method except that a medium containing MUGal10 ^-3 M was used, and the above results were obtained after 10 hours of culture. Example 3 Esherichia coli ATCC25922 at 0.05
% sodium desoxycholate and 10 ^-2 M
The cells were cultured at 37°C for 8 hours using heart infusion broth containing IPTG. This was successively diluted with water to make a suspension of 10 ^-2 to 10 ^-8 cells per ml, centrifuged to collect bacteria, and 4-MUGal was added 3x to this.
Add 5.0 ml of phosphate buffer containing 10 ^-4 M and incubate at 40°C.
It reacted for 1 hour. Next, after adjusting the pH of the reaction solution to 10.5, bacteria were removed, and the fluorescence intensity of the supernatant was measured to determine the amount of 4-MU produced in the reaction solution. Figure 1 shows the relationship between the number of E. coli in the reaction solution and the amount of 4-MU produced.
The vertical axis in Figure 1 is the amount of 4-MU produced (×10 ^-7 M),
The horizontal axis shows the number of bacteria. As shown in Figure 1, the number of bacteria is 4.
-It is well proportional to the amount of MU produced. Urine from 5 patients diagnosed with urinary tract infection
Collect 5.0 ml as usual and add 5.0 ml of heart infusion medium (0.05% desoxycholic acid,
(containing 10M ^-2 IPTG) and held at 37°C for 30 minutes. This is centrifuged to collect the bacterial cells, and this
5.0 ml of phosphate buffer containing 10 ^-4 M of MUβGal and 20 µ of toluene were added and reacted at 40°C for 1 hour.
The amount of 4-MU produced in the reaction solution was measured, and the first
The number of each coliform group was determined based on the calibration curve shown in the figure. On the other hand, the number of coliform bacteria was measured by the conventional method of pouring using a sodium desoxycholate medium, and the results are compared in Table 4.

[Table] Example 4 Escherichia coli (Escherichia coli ATCC10798K-
12) in a broth medium at 37°C for 20 hours, diluted with sterile water in 5 steps (10 to ¹⁰ cells/ml), and each 1.0 ml was diluted with isopropyl-β-D-thiogalactopyranoside ( IPTG) was inoculated into 9.0 ml of bouillon medium containing 10 ^-3 M, and cultured with shaking at 37°C for 1.0 to 6.0 hours. To 20 ml of this culture solution, 20 µ of toluene and 2.0 ml of a phosphate buffer (0.01 M, PH 7.0) containing 3 x 10 ^-4 M 4-MUG were added, and the reaction was carried out at 37°C for 60 minutes. . Add 0.5 ml of 1M glycine buffer with pH 11.0 to each reaction solution, excite it by irradiating it with ultraviolet light with a wavelength of 360 nm, and measure the fluorescence at 450 nm with a fluorometer. The amount of 4-MU produced was measured. The results are shown in Table 5. The numbers of Escherichia coli in Table 5 were determined by culturing for 48 hours by the plate serial dilution method using a bouillon plate agar medium. By utilizing the relationships shown in Table 5, the number of initial bacteria or the number of E. coli in a sample can be measured in a short time. As for the measurement time, even if the initial number of bacteria is 1 per 10 ml of culture medium, detection is possible in a total of 6 hours, including 5 hours of culture and 1 hour of enzymatic reaction.

[Table] Example 5 Heart Infusion Broth (HI-medium), HI medium supplemented with 10 ^-3 IPTG, PTG, or 1.0% lactose (10 ml each)
After inoculating ¹⁰ cells of E. coli ATCC 10798 and culturing them for 5 hours, the number of bacteria in each culture solution was determined.
In each case, there were 1.6×10 ⁶ pieces per 10 ml. The β-galactosidase activity of each culture solution was measured in the same manner as in Example 1, and the results shown in Table 6 were obtained. Table 6 Addition effect of inducer Inducer 4 -MU production amount (M) Control (no addition) 1.5×10 ^-6 IPTG 1.5×10 ^-5 PTG 1.5×10 ^-5 Lactose 8.7×10 ^-6 Next, the above Add 20μ of ethyl acetate to each 2.0ml of culture solution obtained using HI medium plus IPTG, and treat at 35°C for 60 minutes or sonicate (10KH, 10
2.0 ml of MUGal solution (3x
10 ^-3 M, 0.01 M phosphate buffer) and incubated at 37°C for 60 min.
The reaction was carried out for a minute, and the amount of 4-MU produced was measured.
The results are shown in Table 7. Table 7 Method for destroying bacterial cells Method 4 - MU production amount (M) Ultrasonic irradiation 1.5×10 ^-5 Acetic acid addition method 1.5×10 ^-5 Control (no treatment) 1.5×10 ^-7 Table 6 and From the results in Table 7, it is possible to measure if the initial number of bacteria is large even without the addition of β-galactosidase inducers or without extracting the enzyme from the cells, but when IPTG or PTG is added. This increases the sensitivity by approximately 10 times, and also
The addition of ethyl acetate treatment and ultrasonication increases the sensitivity by 100 times, indicating that these operations are effective when the initial number of bacteria is small. Example 6 Five tubes of 10 ml, 1 ml, 0.1 ml, and 0.01 ml of river water were taken and transplanted into 10 ml of heart infusion medium containing 10 ^-3 M IPTG and 0.1% sodium desoxycholate at 37°C for 6 hours. The cells were cultured with shaking, and the supernatant of the culture solution was examined for the presence or absence of fluorescence. The results are shown in Table 8. In addition, the number of coliform bacteria in the same river water was measured based on the serial dilution method (optimal method) stipulated in Environment Agency Notification No. 59, and the results are also listed.

[Table] Used.
Using the results in Table 8 to determine the number of coliform bacteria in 100ml of sample water, it was 79/100ml. As described above, it was confirmed that according to the present invention, coliform bacteria can be detected in 7 hours, and the results are completely consistent with the results measured in 48 hours using the most probable method conventionally used. Example 7 After leaving 100g of commercially available potato korotsuke overnight
Add to 100ml of phosphate buffer (PH7.0, 0.1M),
Homogenize this 1ml, 0.1ml, 0.01ml,
Take 0.001ml and add 0.1ml of sodium desoxycholate.
They were each inoculated into 10 ml of heart infusion broth containing 1.5% and cultured with shaking at 44.5°C for 10 hours. Phosphate buffer ( ^0.01M ,
PH7.0) was added and kept at 37°C for 60 minutes. 0.5 ml of glycine buffer with a pH of 11.0 was added to each reaction solution, and after sterilization, the number of fluorescent supernatants was determined. The same specimen was measured using the conventional method (most probable method). The results are shown in Table 9.

【table】

[Table] When the coliform group in 100 ml of sample was determined using the most probable number table from the results in Table 5, it was confirmed that both methods were in complete agreement with 49 coliform bacteria/g. Example 8 Weighed 10 g of orchard soil, added 90 ml of water to it, homogenized it thoroughly, and added chloromycetin to it.
100r/ml, YM medium containing MUP10 ^-3 M and MUβGal10 ^-4 M (yeast extract 0.05%, malt extract
0.05%, PH6.0) 1.0ml, 0.1ml, 0.01ml each 3
It was added to the original medium and cultured with shaking at 27°C for 12 hours.
After adjusting the pH of the resulting culture solution to 10.5, bacteria were removed and the supernatant was examined for the presence or absence of fluorescence to determine the number of fluorescent cells, and the number of bacteria was determined using the most probable number table. The number of bacteria was 1500. Contain 100 r/ml of chloromycetin in the same sample as usual.
YM agar medium (yeast extract 0.3%, malt extract
0.3%, peptone 0.5%, glucose 1.0%, agar
1.5%, PH6.5) was cultured at 27°C and the number of yeast colonies that appeared was measured and found that 10g of soil
There were 1,300 pieces inside.

[Brief explanation of drawings]

FIG. 1 is a graph showing the relationship between the number of viable Escherichia coli bacteria and the amount of 4-MU produced.

Claims (1)

[Scope of Claims] 1. A test solution containing a certain amount of a specimen or a mixture obtained by culturing the specimen in a nutrient medium for 2 to 12 hours and adding a non-fluorescent umbelliferone derivative to the culture is maintained for a certain period of time, or the specimen is After culturing in a nutrient medium containing a non-fluorescent umbelliferon derivative for 2 to 12 hours, the amount of fluorescent umbelliferon derivative released in the mixture or the nutrient medium is measured, and the microorganisms in the sample are determined based on the amount of the derivative released. A rapid microbial testing method consisting of counting numbers. 2. The non-fluorescent umbelliferone derivatives include 4-methylumbelliferyl phosphate, 4-methylumbelliferyl-α-D-galactoside,
4-Methylumbelliferyl-β-D-galactoside, 4-methylumbelliferyl arabinoside, 4-methylumbelliferyl pyrophosphate, 4-methylumbelliferyl acetate, 4-methylumbelliferyl acetamide
The method for detecting microorganisms according to claim 1, wherein the method is one or more non-fluorescent umbelliferone derivatives selected from the group consisting of β-D-glucopyranoside and 4-methylumbelliferon glucoside. . 3. The method for testing microorganisms according to claim 1 or 2, wherein the microorganisms are common bacteria. 4. The microbial testing method according to claim 1 or 2, wherein the microorganism is coliform bacteria.