JPH01119752A - Polyamine sensor - Google Patents

Abstract

PURPOSE:To enable the simple and highly sensitive measurement of all of putrescine, cadaverine and spermidine in a short time, by a method wherein putrescine oxidase derived from micrococcus rubens is fixed on a carrier to form a fixed enzyme membrane. CONSTITUTION:A fixed enzyme membrane 3 formed by fixing putrescine oxidase derived from micrococcus rubens is fitted to the detecting end of a ground electrode 2. When a sample contacts with the enzyme membrane 3, H2O2 is produced by an enzyme reaction wherein polyamine such as putrescine, spermidine, cadaverine, etc. operates as a substrate, and the quantity of polyamine is measured by detecting said H2O2 by the ground electrode 2. This sensor prepared according to this constitution has an excellent sensitivity to all of putrescine, spermidine and cadaverine, and accordingly the quantity of polyamine can be measured thereby rapidly and accurately.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to a sensor for detecting the amount of polyamines such as putrescine, spermidine, and cadaverine, which increase due to microbial decay of living tissues and abnormal growth in living organisms, and further relates to More specifically, the present invention relates to a polyamine sensor that can quickly detect the amount of polyamine and is effectively used for determining meat freshness, diagnosing cancer, and the like.

[The problem that conventional techniques and inventions are trying to solve] Currently, meat freshness can be determined using the amount of polyamines such as putrescine, spermidine, and cadaverine, which are produced due to microbial decay of biological tissues and abnormal growth within the body. It is proposed to perform a cancer diagnosis.

In other words, as people's eating habits change, the consumption of meat and its processed products is increasing year by year, but the supply of meat is not only domestic but also imported in large quantities from foreign countries. During transportation and distribution, it is delivered to consumers and processing manufacturers in a fresh state by refrigeration or freezing, but the actual determination of freshness is based on experience based on sensual methods such as appearance and smell. is the current situation.

This is a scientific method for determining the freshness of meat.

The most common method is based on the number of microorganisms, and the number of viable bacteria is less than 5 x 10s per gram of meat.
It is considered to be edible if it is 10'' to I x 10', and not edible if it is 1 x 107 or more. However, determination based on viable bacterial count requires at least 48 hours for the result to become clear and is not practical. Various chemical indicators have been proposed, such as porphyrins, but none are conclusive.

On the other hand, in the field of seafood, the value based on the post-mortem decomposition of nucleotides has long been studied as a freshness indicator that indicates the so-called ``liveness'', and in recent years, value measurement sensors or value test strips have been used to measure the quality of seafood. It has come into practical use as a quick and simple means of determining freshness.

By the way, polyamines are one of the substances produced during the process of meat being putrefied by bacteria, and there have been several research reports showing its effectiveness as an indicator for determining the freshness of meat.

Since polyamines are metabolized in living tissues according to the pathway shown in the following formula (1), putrescine, spermidine, and spermine are already detected in meat immediately after layer killing. Immediately thereafter, polyamines are produced and accumulated by bacteria, but this is due to decarboxylation of amino acids that are abundant in meat, and as shown in formula (2) below, polyamines are produced and accumulated. An amine is produced. Among the research reports, Birgit Wor
Tberg etc. are putrescine, cadaverine, histamine,
The results show that it can be judged as fresh when the total amount of tyramine is 5 ppm or less. Also, R, A, E
It has been found that when the number of viable bacteria per 1g or 1cj of dwordskJL±meat becomes 101 or more, the total amount of putrescine and cadaverine begins to increase significantly. Also, in the field of seafood, E, K armas is Bioge.
The following formula is proposed as nic A m1ne index (BΔengineering). In addition, as a recent study, Yamanaka reported that agmatine is effective as a freshness indicator for squid, and cadaverine is effective for red-fleshed fish.

In contrast, the present inventors, as shown in Table 1 below,
Data has been obtained that the total amount of putrescine, cadaverine, and spermidine increases significantly as meat spoils, and that the total amount of putrescine, cadaverine, and spermidine is also effective as an indicator for determining the freshness of meat. The significance of measuring polyamines, which are converted from amino acids by microbial action through the pathway of formula (2) above, has been recognized as an indicator of postmortem decomposition of animal tissues.

Furthermore, polyamines undergo metabolism in vivo as shown in formula (1) above, and are widely present in animal and plant cells. They contribute to the stabilization of cell membranes and at the same time act as a kind of growth factor, and are thought to be involved in the biosynthesis of nucleic acids and proteins.Therefore, they are present in large numbers in tissues where protein metabolism is active, such as the armpits and liver. . Since Russell et al. reported in 1971 that polyamines were found to be high in the urine of cancer patients, polyamines have become l1ff! It has been recognized to have clinical significance as a tumor marker.

In these cases, in order to detect early spoilage of meat, it is necessary to detect whether several ppm of polyamines are present in the same weight of meat extract, as shown in Table 1. For diagnosis, it is necessary to detect polyamines in the order of several tens of μM in urine.

Conventionally, methods for measuring polyamines such as putrescine, cadaverine, and spermidine include chromatographic separation of polyamines in reversed-phase ion pair mode or ion exchange mode, and detection by adding fluorescent reagents to the separation. It is being

However, since this method is based on separation analysis, individual quantification is possible and the data is highly reliable, but it requires expensive equipment to perform high-performance liquid chromatography, and it is difficult to obtain results. The disadvantage is that it takes 20 to 30 minutes to measure, and rapid measurement cannot be performed.

Furthermore, it is beneficial to utilize various amine oxidases for the purpose of selectively measuring polyamines.

This enzyme has many known origins, each with its own unique substrate specificity. Details of this are described by Kumagai, Yamada et al. in ``Proteins, Nucleic Acids, Enzymes J vol, 29 (1984) p. 357-373.

In this case, putrescine oxidase derived from Micrococcus rubens is said to be effective for measuring urinary polyamines, which is effective in cancer diagnosis and curative management, and a liquid reagent containing this putrescine oxidase is used. A measurement kit for measuring polyamines in urine by an end point method (terminal method) in combination with various coloring reagents has already been put into practical use.

However, measurement using the above-mentioned endpoint method requires complete decomposition of the substrate, which requires a considerable amount of time to wait for the reaction to reach the end point, and has the disadvantage that rapid measurement cannot be performed. For example, the Polyamine Test Enzyme sold by Ginotest Co., Ltd. is designed to perform enzyme-based assays alone for 15 minutes at 37°C. In addition, putrescine oxidase shows activity against a wide range of polyamines such as putrescine, cadaverine, and spermidine, but their specific activities are not the same, and when putrescine is taken as 100, the relative specific activity is cadaverine 7.2. Spermidine is said to be about 10, (Agri, Biol, Ch
Em, 30 1202 (1966)), therefore, when measurements are performed using the late assay method, there is a problem of low sensitivity to cadaverine and spermidine.6 Furthermore, both the end point method and the late assay method require expensive spectrophotometry. In addition to the need to use equipment to read the results, reagents containing enzymes are consumed each time a measurement is performed, resulting in high running costs.

For this reason, there has been a demand for a polyamine 411I determination device that can be easily and repeatedly used in the field.

The present invention was made in view of the above circumstances, and is a polyamine that can easily and quickly measure all of putrescine, cadaverine, and spermidine with high sensitivity, and can be suitably used for determining meat freshness, diagnosing cancer, etc. The first objective is to provide a sensor.

[Means and effects for solving the problem] As a result of intensive studies to achieve the above object, the present inventors immobilized putrescine oxidase derived from Micrococcus rubens on a carrier to form an immobilized enzyme membrane. It has been found that, when immobilized by a covalent bonding method, the sensitivity of putrescine oxidase to spermidine and cadaverine is significantly higher than that of natural humoral enzymes.

In other words, the so-called oxygen electrode, in which an immobilized enzyme membrane on which an enzyme is immobilized is attached to a base electrode such as an oxygen electrode or a hydrogen peroxide electrode, is a type of initial rate modified method (late assay) for the use of enzymes, and is a short and short method. It has the advantage of being able to perform measurements over time and of being able to use the enzyme repeatedly, but the sensitivity of the measurement generally decreases, and especially for substrates with low relative specific activity, there is a tendency for the sensitivity to decrease further. In response to this, the present inventors found that when an immobilized enzyme membrane was created using putrescine oxidase derived from Micrococcus rubens, the apparent output sensitivity to spermidine and cadaverine was surprisingly improved compared to that of a humoral enzyme. Therefore, putrescine can be immobilized by using an oxygen electrode with the enzyme membrane described above.

The inventors have discovered that the total amount of cadaverine and spermidine can be accurately measured in a short period of time, leading to the present invention.

Accordingly, one object of the present invention is to provide a polyamine sensor comprising an immobilized enzyme membrane on which putrescine oxidase derived from Micrococcus rubens is attached to the detection end of a base electrode.

The polyamine sensor of the present invention has the above-described configuration, so that when the sample comes into contact with the immobilized enzyme membrane, lI202 is generated due to unreacted U with polyamines such as putrescine, spermidine, and cadaverine as substrates, and this l-I2
The amount of polyamine is measured by detecting 02 with a base electrode, and the sensor of the present invention has good sensitivity for all of putrescine, spermidine, and cadaverine, and therefore can quickly and accurately measure the amount of polyamine. It is possible.

In this case 1, although there are no limitations on the means for producing the immobilized enzyme membrane of the present invention, putrescine oxidase derived from Micrococcus rubens is placed on a membrane carrier such as acetylcellulose for producing an immobilized enzyme membrane, and a suitable surface is applied to the surface of the carrier. After the treatment, it is preferable to immobilize by covalent bonding using surface functional groups. Note that the means for immobilizing by covalent bonding is not limited, and various known methods such as the method described in JP-A-62-85853 can be used.

In addition, although there are restrictions on the type of base electrode, enzyme reaction
It is preferable to use an oxygen electrode or a hydrogen peroxide electrode that reflects the wide initial velocity characteristics of .

EXAMPLES Next, the present invention will be specifically explained with reference to examples, but the present invention is not limited to the following examples.

231 Figure 1 shows one embodiment of the present invention, and this polyamine sensor 1 has an oxygen electrode or a hydrogen peroxide electrode (base electrode).
An immobilized enzyme membrane 3 on which putrescine oxidase derived from Micrococcus rubens is immobilized is mounted on the septum 2 (detection end).

FIGS. 2(A) to 2(C) each show other embodiments of the present invention, and these polyamine sensors 1 are mounted on a diaphragm (detection end) of an oxygen electrode or a hydrogen peroxide electrode (base electrode) 2. An immobilized enzyme membrane 3 similar to the above is attached, and an air discharge nozzle 5 is attached to the tip of the air introduction pipe 4.
A cleaning device 6 for cleaning the surface of the enzyme membrane 3 by bubbling air onto the surface of the enzyme membrane 3 from the nozzle 4 is disposed on the base electrode 2.

In each of the above Examples, putrescine oxidase derived from Micrococcus rubens was used as the immobilized enzyme membrane on the surface of a membranous carrier for producing an immobilized enzyme membrane made of acetyl cellulose as disclosed in Japanese Patent Application Laid-open No. 85853/1983. It was immobilized by a covalent bond method using the method described above. In addition, the enzyme membrane prepared in this way is cut into a circular shape of 2φ to 8φ to match the outer diameter of the sensing electrode of the base electrode, and then cut into a circle with a net holder or the like. Installed it on IH4.

Next, we investigated the basic characteristics of the polyamine sensor of the above example and the stability over time of the enzyme membrane, and also conducted measurements at the actual liquid level and sample test 1.Basic characteristics of the polyamine sensor FIGS. 3(A) to 3(C) show calibration curves for each polyamine of the polyamine sensor having the configuration shown in FIG. In addition, the third
In the figure, (A) is spermidine, (B) is putrescine, (C
) is the calibration curve for cadaverine.

As is clear from FIG. 3, spermidine has the highest activity per unit molar concentration, followed by putrescine and cadaverine. Humoral putrescine oxidase has the highest activity against putrescine, and it is
It is known that cadaverine is about 7.2 and spermidine is about 10. From the above results, it is recognized that the effect of a kind of chemical modification on the enzyme by immobilization treatment is manifested.

In addition, the pH characteristics of this polyamine sensor are shown in Figure 4. From the results shown in Figure 4, the sensor of the present invention has the highest output at pII 9.2 for any polyamine; By performing measurements at
Sensitive to the following degrees.

It has been found that it can be used as a practical polyamine sensor for detecting decay.

Next, a polyamine sensor with a configuration as shown in FIG.
Figure 5 (A) shows the calibration curve for resin and spermidine.
Shown in (B). In addition, (A) in FIG. 5 is spermidine,
(B) is a calibration curve for putrescine. In this case as well, it is recognized that the same effects as above are exhibited.

-・Level clause When detecting the actual freshness of meat, 5 to l.

Collect 1 g of meat, add the same weight of pure water and stir with a homogenizer, and use the filtrate or the liquid obtained by soaking the meat in 1 (amount of salt water) and extracting the surface as the sample liquid. The concentration of polyamine in the sample solution obtained was the first
As shown in the table.

An actual side chamber method for detecting polyamines in sample liquids is shown below. The polyamine sensor used had a configuration as shown in FIG. 2, in which an oxygen electrode, which has poor output reproducibility and stability in batch measurements, was used as a base electrode.

ptI9.2 borate buffer 1 in a 100d beaker
Insert 0 mQ, dip the tip of the polyamine sensor, and take the zero point of the electrode output. Add 5 mQ of a solution of putrescine and spermidine at a concentration of 0 to 5 .mu./Q as a standard solution. The calibration curve is as shown in Figure 6, and has additivity to the calibration curve of putrescine and spermidine alone.

Next, when 30 μ/Ω of spermine coexists, the calibration curve becomes as shown in FIG. This is spermine putrescine,
This is because it causes competitive inhibition against spermidine.

As a model that is close to the actual solution, let us assume that a solution containing 30 mg/Q of spermine is a fresh solution, and a solution containing 30 μ/Q of spermine, putrescine, and spermidine as an initial spoilage fluid is used as the initial spoiled fluid. Figure 8 shows the output for each. . The sensor thus shows a clear response to decay.

Sample test Transfer frozen pork to a refrigerator at 4℃, homogenize the meat after 80 minutes with the same weight of pure water, filter the liquid, and use 5 mQ.
The results of sampling and subjecting to the polyamine sensor are shown in the 9th section.
As shown in the figure. In addition, meat that has been refrigerated after thawing can be
The sample was obtained by slicing and extracting 20 oz of the sample by shaking with 20 IIIQ physiological saline. FIG. 10 shows the number of viable bacteria in this sample and the results of sampling 2.5 mQ of the sample and subjecting it to a polyamine sensor.

The sensor showed a clear rise from the 60th mark, and the output gradually increased, corresponding to an increase in the number of viable bacteria. In this way, the polyamine sensor has a viable bacterial count I x 107/
It has been shown that it is possible to detect initial decomposition on the order of g.

Stability of the Electrode Store the polyamine sensor at room temperature and adjust the final concentration from time to time to 10
■The time-dependent change in output when subjected to putrescine of /Q is shown in the first graph.
Shown in Figure 1. The results show that the polyamine sensor has practical stability and can withstand repeated use. Furthermore, when stored in a refrigerator, it was stable for several months as shown in Figure 12.

As described above, the polyamine sensor using an enzyme membrane immobilized with putrescine oxidase derived from Micrococcus rubens has a large output for spermidine and force davelin, and the sample solution is 3aN in which the sensor is immersed.
The practicality of the polyamine sensor of the present invention was confirmed by simply adding it to the liquid, allowing the presence or absence of initial spoilage to be determined in a short time. In this case, the sensor of the present invention can be used repeatedly and its running cost is low.

In the above embodiment, the polyamine sensor was applied to livestock meat, but it can also be applied to other samples containing putrescine, spermidine, and cadaverine.

Furthermore, it is also applicable to polyamines in the form of acetyl conjugates in urine, or to samples that have been hydrolyzed and deconjugated with an acyl polyamine hydrolase or the like.

Furthermore, as is clear from the pII characteristics in Figure 4, the pH
Since it is most sensitive to putrescine, spermidine, and cadaverine at pH 9.2, it can generally be used with this pII, but it loses almost all sensitivity to spermidine and cadaverine at pH 6.8, so if you want to detect only putrescine. is possible by measuring at pH 6.8.

〔Effect of the invention〕

- As explained above, the polyamine sensor of the present invention is
It can quickly and accurately measure the amount of polyamines such as putrescine, spermidine, and cadaverine, and can be easily used for determining meat freshness, diagnosing cancer, etc.

[Brief explanation of the drawing]

FIG. 1 is a schematic front view showing one embodiment of the present invention, and FIG. 2 (
A), (B), and (C) are schematic front views showing other embodiments of the present invention, and FIGS. 3(A), (I3), and (C) are respectively the base Tl! A graph showing an example of the calibration curve of the polyamine of the present invention using an oxygen electrode as a pole, FIG. 4 is a graph showing the pH characteristics of the polyamine sensor of the present invention, FIG. 5 (A)
, (B) are graphs showing an example of the calibration curve of the polyamine sensor of the present invention using a hydrogen peroxide electrode as the base electrode, and Figures 6 and 7 are graphs showing an example of the calibration curve of the polyamine sensor of the present invention using an oxygen electrode as the base electrode. 1 liter under actual liquid level conditions
Fig. 8 is a graph showing an example of the output of the polyamine sensor of the present invention in measurement under actual liquid level conditions, and Figs. 9 and 10 are graphs showing an example of the calibration curve in the sample test. A graph showing an example of the output of the polyamine sensor of the present invention when measured, and FIGS. 11 and 12 are graphs showing changes in output over time when the polyamine sensor of the present invention is stored, respectively. Applicant Food Industry Online Sensor Technology Research Association Representative Patent Attorney Takashi Kojima Figure 1 Figure 2 Figure 5 (A) 0 +s +L Isa-mi;-;JLKtL (-2/zn(B) O-body g IL lo(,, t, > ”, t3; f,, rf <-3/
l) Ogog Ibushifu''kai > 1/1 Ko-lman Fig. 7 O+ Zanabu', knee 111 Shin LL (Go/J! N Fig. 8
Figure 9 Figure 10 Figure 11 OIQ λo jo
Shun Thl;8eL(E2)<pg'?
x+:1lhEFigure 12

Claims (1)

[Claims] 1. A polyamine sensor comprising an immobilized enzyme membrane on which putrescine oxidase derived from Micrococcus rubens is attached to the detection end of a base electrode. 2. The polyamine sensor according to claim 1, wherein the immobilized enzyme membrane is one in which putrescine oxidase derived from Micrococcus rubens is immobilized on a carrier by a covalent bonding method. 3. The polyamine sensor according to claim 1 or 2, wherein the base electrode is an oxygen electrode or a hydrogen peroxide electrode.