JP3120505B2 - Aerobic microorganism measuring method and measuring device - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for measuring aerobic microorganisms and a measuring apparatus used for the method. For more information,
The present invention relates to a method and apparatus for quickly measuring the viable cell count (activity) of aerobic microorganisms.

[0002]

2. Description of the Related Art Measuring the concentration of living microorganisms suspended in water is important in handling microorganisms. For example, for controlling a fermentation production process or controlling the quality of fresh food products, a quick method for measuring the concentration of viable bacteria is required.

[0003] The most common method for measuring the concentration of viable bacteria is the colony counting method. In this method, a fixed amount of a sample is aseptically cultured on an agar medium, and the number of colonies formed is visually counted, and the viable cell count is calculated based on the number of colonies. As another measurement method, there is a method in which microbial cells are separated from a sample, stained by adding a dye solution thereto, and the number of viable cells stained is measured under a microscope.

[0004]

However, in the colony counting method, a sample is diluted at a constant dilution rate under aseptic conditions, and cultured at rest for at least 24 hours, and the number of generated colonies is counted with the naked eye. For this reason, a great deal of time and labor is required, and a very complicated operation is required to be skilled, and the accuracy obtained is not sufficient. On the other hand, in the staining method, dead sterilized bodies contained in the sample, various solids in the medium, etc. are stained in the same manner as living microorganisms, so it is extremely difficult to accurately measure the number of viable microorganisms. is there.

[0005] For this reason, a method has been proposed in which the catalase activity of yeast or bacteria is measured, and the number of viable bacteria such as bacteria is calculated based on the measured value (Japanese Patent Publication No. 55-15999, US Patent No. 3838034). No.), but it is necessary to separate bacteria etc. from the sample, and the setting of enzyme reaction conditions is complicated.
Again, quick measurements cannot be made.

[0006] As described above, none of the conventional methods can measure the viable cell count quickly and with high accuracy, and can quickly control the viable cell count concentration by controlling the production process using microorganisms and controlling the quality of foodstuffs. It could not be applied to areas that required measurement.

The present inventor has studied to improve such a conventional viable cell counting method, and has previously proposed a viable cell concentration measuring apparatus using an insoluble pyridinium type resin and a diaphragm type oxygen electrode ( Japanese Patent Application No. 1-300633). However, this apparatus has a drawback that the operation of attaching the resin beads to the oxygen electrode requires skill, and the measurement results vary widely.

An object of the present invention is to provide a method and an apparatus for measuring the viable cell count of aerobic and facultative anaerobic microorganisms suspended in water extremely quickly and accurately by a simple operation. .

[0009]

The inventor of the present invention has found that a nonwoven fabric coated with an insoluble pyridinium-type resin can strongly capture microorganisms in a living state, and can reduce the oxygen consumption of microorganisms captured by this immobilization method. The present inventors have found that there is a close relationship between the concentration of viable bacteria and have completed the present invention. The present invention arranges a non-woven fabric coated with a water-insoluble pyridinium-type resin in a sensitive portion of an oxygen electrode, contacts a sample solution containing microorganisms with the non-woven fabric, based on a change in the detection output of the oxygen electrode at the time of contact. And a method for measuring the number of viable aerobic microorganisms in the sample solution. Further, the present invention provides a sensitive part of the oxygen electrode,
An object of the present invention is to provide an apparatus for measuring aerobic microorganisms equipped with a flow cell having a nonwoven fabric coated with a water-insoluble pyridinium-type resin.

[0010] The measuring device of the present invention is an electrochemical sensor for rapidly measuring the viable cell concentration of aerobic and facultative anaerobic microorganisms in an aqueous suspension (floating liquid), and comprises an oxygen electrode and a pyridinium-type polymer. Some N-benzyl-4
-Consisting of a flow cell comprising a nonwoven fabric coated with a copolymer of vinylpyridinium and styrene. When a suspension sample of the microorganism passes through the electrochemical sensor at a constant flow rate, the microorganism is captured and accumulated on the nonwoven fabric in a living state. Oxygen consumption by the captured live bacteria is detected by the oxygen electrode as a decrease in current, and the rate of current decrease is proportional to the concentration of live bacteria.

The insoluble pyridinium type resin used in the present invention means a water-insoluble resin having a pyridinium group in a polymer chain. Preferred examples of the insoluble pyridinium-type resin include a vinyl copolymer having the following formula [Chemical Formula 1] and the following formula [Chemical Formula 2] as a structural unit, or a polymer having the following Formula [Chemical Formula 1] as a structural unit. A crosslinked vinyl polymer crosslinked by a polymer chain having the following formula [Formula 2] as a constitutional unit is exemplified.

[0012]

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[0013]

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Wherein R ₁ is a benzyl group, having 4 to 16 carbon atoms.
R ₂ is an alkyl group having 1 to 3 carbon atoms, X is a halogen atom, Y is a hydrogen atom, an alkyl group having 1 to 3 carbon atoms, a benzyl group, an aryl group, an ether group, a carboxyl group, Or a carboxylic acid ester group] The molar ratio of the structural units [Chemical Formula 1] and [Chemical Formula 2] is not particularly limited. However, in order to efficiently capture microorganisms while maintaining water-insolubility, the general formula [Chemical Formula 1]: 2] = 1: 20 to 1: 0.0
It is preferably 1. The degree of polymerization is usually sufficient if it is 100 or more. In addition, the crosslinked product may be less than this.

The insoluble pyridinium-type resin can be synthesized from the corresponding monomer by a known polymerization method or a crosslinking reaction, and various commercially available products such as powder, granule, film, and fiber can be easily obtained. sell.

A conventionally known nonwoven fabric may be used as a base material for coating the resin. Further, as the oxygen electrode used in the present invention, a so-called diaphragm type oxygen electrode is preferable.

In the measurement method of the present invention, a device having a non-woven fabric coated with the above-mentioned water-insoluble pyridinium-type resin attached to the sensitive part of the oxygen electrode is usually used,
It is preferable to carry out the sample by circulating the sample in the flow cell for a certain period of time. The circulation time at this time is sufficient to be 10 to 30 minutes, and the aerated sample may be introduced into the flow cell.

According to the present invention, the viable cell count (activity) of various aerobic microorganisms present in a sample can be quickly measured based on the output of the oxygen electrode. In the present specification, aerobic microorganisms mean aerobic microorganisms in a broad sense that can consume oxygen during the growth process, and include so-called facultative anaerobic microorganisms and arbitrary anaerobic microorganisms, and do not consume oxygen at all. Excluding anaerobic microorganisms.

[0019]

When a suspension of microorganisms is injected, the microorganisms in the suspension are trapped on the surface of the nonwoven fabric, and if the trapped microorganisms are alive, they consume oxygen, so that the oxygen concentration decreases. If a suspension of a constant concentration is continuously flowed at a constant speed, the amount of captured microorganisms increases in proportion to the flow time, and the current value decreases at a constant speed. When the concentration of microorganisms in the suspension is high, the amount of microorganisms captured per unit time is large, so that the current reduction rate is high. If the relationship between the suspension concentration and the current reduction rate is determined in advance for a specific microorganism, the concentration of microorganisms contained in an unknown suspension can be determined from the current reduction rate. Insoluble pyridinium-type resins also capture dead microbial cells, but do not consume oxygen and do not cause a decrease in current. In this way, the number of viable bacteria (activity) in the sample can be immediately known from the decrease in the current value, and rapid measurement can be performed by a simple operation.

[0020]

Next, the present invention will be described more specifically based on examples. FIG. 1 is a schematic sectional view of the sensor of the present invention. In FIG. 1, an aerobic microorganism measuring apparatus 1 of the present invention is shown.
Consists of an oxygen electrode (electrochemical meter 7616 type) 10, a flow cell 20 disposed in a sensitive portion thereof, and an insoluble pyridinium-type resin-coated nonwoven fabric 30 disposed therebetween.

The oxygen electrode 10 may be a known one,
It consists of a Teflon membrane (50 μm thickness), a platinum cathode, an aluminum anode and a saturated potassium chloride electrode.

The flow cell 20 is attached to the upper flow cell 21 and a similar lower flow cell 22 of a pipe made of polymethyl methacrylate (inner diameter 8.0 mm, outer diameter 12 mm), and the upper and lower flow cells are attached to the outer periphery of a connection portion thereof. And a silicon pipe (inner diameter: 10 mm, outer diameter: 14 mm) 23 connected to the main body. The lower flow cell 22 is provided with a test liquid introduction pipe 24 extending from the outside to the upper end of the lower flow cell, and a drainage pipe 25 discharging the test liquid from a position higher than the upper end of the lower flow cell 22 to the outside. The introduction pipe 24 and the drain pipe 25 are made of a stainless steel pipe (inner diameter 1.0 mm, outer diameter 2.0 mm) and are sealed and fixed to the lower flow cell 22 by an epoxy adhesive 26. A nylon mesh (100 mesh) 27 is provided at the upper end of the upper flow cell 21.

When the flow cell 20 is attached to the sensitive part of the oxygen electrode 10, a nonwoven fabric 30 coated with an insoluble pyridinium resin is sandwiched between them and fixed by the auxiliary device 2.

The non-woven fabric used here is a non-woven fabric made of pure rayon WE-8890 (a fineness of 1.5 denier and a thickness of 0.5 m).
m, surface area 2020 cm ² / g; Japan Vilene Co., Ltd.
Made). This nonwoven fabric was coated with a 1: 2.75 (molar ratio) copolymer of N-benzyl-4-vinylpyridinium chloride, which is a pyridinium-type polymer, and styrene (hereinafter, referred to as BVPS). The coating was performed by dipping the nonwoven fabric in a 0.5% polymer solution (solvent; mixed solvent of methanol: acetone = 6: 4) overnight (coating amount: 10 mg / g). BVPS contains 2.94 mg / g of a pyridinium group and has an intrinsic viscosity of 0.23 dl / g (10 g / MgCl ₂ .6H ₂ O).
l in ethanol at 30 ° C).

A measuring system (constant temperature at 25 ° C.) shown in FIG. 2 in FIG.
1 denotes an air pump for aeration, 42 denotes a peristaltic pump (peristaltic pump), 43 denotes a reference liquid or sample liquid tank, and 44 denotes a recorder. Sample suspension of test microorganism and pH 7.0
0.05M sterile phosphate buffer (standard solution, hereinafter referred to as PBS)
) Is guided to the flow cell of the measuring device 1 at a constant flow rate by a peristaltic pump (Tokyo Rika Instruments Ira Micro Pump MP-3 type) 42, and a recorder (Rika Denki R-21)
(Type) 44 and record the current.

In the measurement using the apparatus of the present invention,
Air and PB at 5 ° C, 4 ml / min
The liquid S was allowed to flow until the current value of the recorder 44 became constant.
Then, when the reference PBS solution is switched to a PBS suspension of the test microorganism and the solution is flowed at 25 ° C., pH 7.0 and a flow rate of 4.0 ml / min, the current value decreases with time. The rate of decrease of the current value was evaluated in nA / h. This current reduction rate was recorded and compared with the turbidity of the microbial cell suspension. Since the capture of microbial cells by the non-woven cloth impregnated with the insoluble pyridinium-type resin is very powerful and irreversible, it is preferable to replace the resin for each measurement.

(Test microorganism) (1) Achromobacter polymorph
polymorph) ICR (Kyoto University Institute for Chemical Research) B
-00880, (2) Alcaligenes faecalis (Al
caligenes faecalis) IFO13111, (3) Alcaligenes faecalis IAM
(Collection number of Institute of Applied Microbiology, The University of Tokyo) b-141
1, (4) Bacillus subtilis
IFO3037, (5) Enterobacter cloacae IFO3320, (6) Escherichia coli BICRB-00
120 strains, (7) Pseudomonas aeruginosa (Ps
eudomonas aeruginosa) IFO3080, (8) Serratia marcescens IFO30
46, (9) Staphylococcus aleus (Staphy
lococus aureus) IFO3060, (10) Debaryomyces hansenii IFO
0023, (11) Saccharomyces Sembiche (Sa
ccharomyces cerevisiae) IFO1662

A. Polymorph, A. E. faecalis and S. f. Marcesense was cultured at 30 ° C. for 4 hours. B.
Subtilis, E. Black fly, E. Coli, p. Aeruginosa and S.A. Aureus was cultured at 37 ° C. for 4 hours.
D. Hansenii and S.M. Cereviche was cultured at 28 ° C. for 4 hours. A. Polymorph, A. E. faecalis, B. Subtilis, E. Black fly, E. Coli, p. Aeruginosa, S.A. Aureus and s. The nutrient broth used for culturing Marcesense was 5.0 g of peptone and 3.
It was prepared by dissolving 0 g in 1000 ml of water having a pH of 7.2. D. Hansenii and S.M. The medium used for cultivation of S. cerevisiae was 10.0 g of glucose and 3.0 g of yeast extract.
g in 1000 ml of water at pH 6.0.
These microbial cells were collected by centrifugation at 2000 × g for 30 minutes. The collected cells were washed three times with PBS, and suspended in PBS under aseptic conditions. Using these suspensions, live cell concentration measurements and turbidity measurements at 610 nm were performed. Since the measurement was performed immediately after preparation of the microbial suspension, cell damage before the measurement would be negligible.

In the measurement, PBS was used until the current became stable.
For a short time through the sensor system. When a sample suspension of test microbial cells was passed through the sensor system at a constant flow rate, the current was initially slightly disturbed and then showed a uniform decrease. It took about 10 minutes per measurement for reliable confirmation of the current reduction rate.

Test Example 1 FIG. 3 is a chart showing a decrease in the current value of the oxygen electrode when an A. faecalis suspension having an absorbance of 0.4 at 610 nm was injected into the apparatus of the present invention. The current decreasing rate was 160 nA / h.
In addition, E. coli (optical density of 0.1 at 610 nm) autoclaved (pressure steam sterilization) at 121 ° C. for 20 minutes.
When the dead cell suspension of 1) was passed through a flow cell, the decrease in current was negligible. Therefore, the electrochemical sensor of the present invention records only living cells.

FIG. 4 is a chart showing the relationship between the current reduction rate and the microbial cell concentration. A linear relationship is obtained between the current reduction rate and the optical density at 610 nm, where the current reduction rate is proportional to the microbial cell concentration in the sample suspension. Since the method most frequently used to evaluate the concentration of microbial cells is the turbidity method, the relationship between the current reduction rate and the optical density at 610 nm was examined. Colony counting is particularly important for viable cell concentration measurements, but the quantitative accuracy of this measurement is not sufficient as desired. The electrochemical method of the present invention comprises:
Since the microbial cell suspension was performed immediately after preparation, cell damage and decrease in viability before measurement would be negligible.

Using the measurement method of the present invention, aerobic bacteria 3
The strain was examined using 5 strains of facultative anaerobic bacteria and 2 types of yeast. The ratio between the rate of current decrease and the turbidity of the microbial cell suspension at 610 nm is highly dependent on the type of microorganism. Table 1 shows the results. These results show that the current reduction rate is a useful index of the live microbial cell concentration.

The minimum detectable concentration of viable cells by the electrochemical measurement method of the present invention is defined as the optical density at 610 nm of 0.1.
Was evaluated as a turbidity equivalent concentration corresponding to three times the standard deviation using a microbial cell suspension in PBS. The results are shown in Table 1. The minimum detectable concentration is 0.005 to 0.00 as turbidity.
023, corresponding to a bacterial concentration on the order of 10 ⁷ cells / ml.

[0034]

[Table 1]

As described above, according to the measuring method of the present invention, the concentration of viable cells can be measured quickly and the microbial cells based on the conventional total cell carbon analysis for measuring the total concentration of microorganisms regardless of the viability of the cells. The concentration measurement method is complemented.

The temperature and pH with respect to the current decrease rate
5 and 6 show the results obtained by examining the effect of. The rate of current decrease increased somewhat above 30 ° C. (FIG. 5).
The effect of pH on the rate of current decrease was not significant (FIG. 6).

[Comparative Test Example] When an E. coli suspension having an optical density of 0.2 at 610 nm was measured using BVPS resin beads instead of the BVPS-coated nonwoven fabric, the current varied widely as shown in FIG. The sensitivity of the sensor system was low. The minimum detectable turbidity concentration evaluated as a concentration equivalent to three times the standard deviation using various microbial cell suspensions in PBS having an optical density of 0.1 at 610 nm is shown in Table 2. It was in the range of 0.06-0.29.

[0038]

[Table 2]

As described above, the minimum detected turbidity concentration when the BVPS-coated nonwoven fabric is used is in the range of 0.005 to 0.023. The reason for the relatively low sensitivity and large current fluctuations in the case of BVPS resin beads is that the sample suspension passes through the resin bead layer with less uniform flow as it passes through the nonwoven fabric. It is thought that there is a cause. The average particle size of the BVPS resin beads was 0.3 to 0.4 mm in a mixed state. The size of the crack in the resin bead packed layer is much larger than that of the non-woven fabric, and as a result,
It is believed that the flow of the microbial sample suspension becomes more uneven and the sensitivity of the sensor system is reduced. Thus, the electrochemical sensor system includes BV, an insoluble pyridinium-type resin.
Nonwoven fabrics coated with BVPS, a pyridinium-type polymer, are more suitable than PS resin beads.

[0040]

According to the measuring method and apparatus of the present invention, the number of viable aerobic and facultative anaerobic microorganisms suspended in water can be measured very quickly and accurately by a simple operation. it can. Therefore, the measurement method of the present invention is useful for controlling a production process using a microorganism and controlling the quality of food.

[Brief description of the drawings]

FIG. 1 is a schematic sectional view showing a measuring apparatus of the present invention.

FIG. 2 is a schematic explanatory view showing a measurement system for performing the measurement method of the present invention.

FIG. 3 shows an A.D. having an optical density of 0.4 at 610 nm. 5 is a chart showing a change in current when the E. faecalis suspension is passed through the sensor at 25 ° C. and a flow rate of 4.0 ml / min.

FIG. 4. A. in PBS. It is a graph which shows the relationship between the electric current reduction rate in a fecalis suspension, and the optical density in 610 nm.

FIG. 5: Optical density 0.2 at 610 nm, pH 7.0
4 is a graph showing the effect of temperature on the current reduction rate of E. coli suspension in PBS.

FIG. 6: Optical density 0.2 at 610 nm, temperature 25
P for current reduction rate of E. coli suspension in PBS
6 is a graph showing the effect of H.

FIG. 7 is a reaction curve of an electrochemical sensor using BVPS resin beads. Optical density 0.2 at 610 nm
Was passed through the sensor at 25 ° C. at a flow rate of 4.0 ml / min.

[Explanation of symbols]

 1: Measuring device 2: Auxiliary device 10: Oxygen electrode 20: Flow cell 21: Upper flow cell 22: Lower flow cell 23: Silicon pipe 24: Introducing pipe 25: Drainage pipe 26: Epoxy adhesive 27: Nylon mesh 30: Non-woven fabric 41 : Air pump 42: Peristaltic pump 43: Reference liquid or sample liquid tank 44: Recorder

────────────────────────────────────────────────── ─── Continued on the front page (58) Fields surveyed (Int. Cl. ⁷ , DB name) C12Q 1/00 C12M 1/34 G01N 27/416 BIOSIS (DIALOG)

Claims (2)

(57) [Claims] 1. A nonwoven fabric coated with a water-insoluble pyridinium-type resin is disposed on a sensitive portion of an oxygen electrode, and a sample solution containing microorganisms is brought into contact with the nonwoven fabric. A method for measuring the number of viable aerobic microorganisms in the sample liquid based on the measurement. 2. An aerobic microorganism measuring apparatus in which a flow cell having a nonwoven fabric coated with a water-insoluble pyridinium-type resin is mounted on a sensitive portion of an oxygen electrode.