JPS584304B2 - Aflatoxine information - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for detecting aflatoxin, and more particularly to a method for qualitative and simple quantitative analysis of aflatoxin in a sample using ascending adsorption chromatography.

Aflatoxin (hereinafter abbreviated as Af) is a type of mold toxin that was discovered in 1960 due to the mass mortality of turkeys in the UK.
Mycotoxin is a general term for polyketide-based metabolites produced by molds (eg, A. flavus) of the genus Aspergillus.

Today, this thing includes B1, B2, B2a, B3, G1,
A total of 10 related and homologous substances have been distinguished, including G2, G2a, GM1, M1 and M2, but B1 has attracted the most attention.

This product accounts for most of the Af species actually produced, and is highly toxic (LD50 = 28 against white Chinese ducklings).
．． 2 μg), and the fact that it has an extremely strong carcinogenic effect is the reason why it is important in public health.

In other words, it has been confirmed that Af B1 has surprising carcinogenicity, causing 100% liver cancer in test mice within one year even at a trace amount of 0.15 to 0.30 μg/day. This carcinogenicity is far stronger than other known carcinogens.

In addition, Af is generally a relatively stable substance that is not easily destroyed at cooking temperatures, and is also biochemically stable, so it remains in the body of the animal that feeds it or is excreted from the body through milk. Therefore, it is extremely dangerous to eat meat or drink milk from animals contaminated with Af.

In particular, Aspergillus oryzae that produces Af is a common mold,
Considering the fact that Af-contaminated grains and beans tend to grow on grains and beans, which are important foods (feed) for people and households, it can be said that there is an extremely high risk of ingesting food contaminated with Af.

Based on the above, much research has been carried out on methods for analyzing aflatoxins present in grains, beans, or their processed products, and an official method has already been established for peanuts and their products (1971). March 16th
(See Attachment (1) of Nikkan Eclipse No. 128), this official method requires a considerable amount of time and effort (approximately 8 hours per sample) for extraction and purification of aflatoxins in samples, and preparation of plates for thin layer chromatography. In addition, special equipment such as a rotary evaporator is required, and the analysis technique itself requires considerable skill, so it is not something that can be carried out easily by those who desire analysis.

This has become a serious problem for those who frequently handle large amounts of grains, pulses, etc. to be tested.

Thus, it can be said that the development of a simple detection method capable of easily detecting or quantifying Af without requiring special equipment or techniques was an important challenge for those skilled in the art.

By the way, the one that seems to be the most practical among the simple Af analysis methods that have been published so far was developed by C.E. Holladay.
This is the millicolumn method by Mr. Ay) (J. Am. Oil
Chem. Soc. Vol. 45, pp. 680-682 (1
968)).

This method uses a milli-column filled with silica gel in a glass tube with an inner diameter of 4 m/mφ.The milli-column is immersed in a sample extract of a chloroform:methanol (97:3) mixture, developed, and dried. The purpose is to detect and easily quantify Af by irradiating it with long-wavelength ultraviolet rays and determining the presence or absence and intensity of a blue-green fluorescent band unique to Af.

Although the original report states that it is possible to measure up to a concentration of 5 ppb (5X10-9), additional tests by the inventors have revealed that in actual samples, various components in the raw materials and mold pigments interfere with detection. The critical concentration was found to be at most 30 ppb.

Since the permissible limit for Af B1 in Japan is 10 ppb, it is difficult to put Holladay's simple method into practical use.

However, as a result of repeated research aimed at improving the detection limit of the millicolumn method, the present inventors found that when dealing with samples with many impurities, Holladay's method using a single adsorption layer does not allow the sample extract itself to be used. It is difficult to separate impurities from Af unless it is sufficiently purified. However, when the adsorption layer is configured as a multilayer structure with relatively different adsorption capacities for Af, even if the sample extract is insufficiently purified, it is difficult to separate the impurities from Af. It has been discovered that the performance of this method is improved, and that the separation by this multilayer adsorption layer can be made more complete by appropriately performing secondary expansion.

In the first place, as long as we aim to establish a simple method, it is desirable to avoid the troublesome purification of the extract as much as possible, so the above discovery is important for this purpose.

The present invention has been developed based on the above knowledge, and an important gist thereof is the use of a special multilayer millicolumn.

FIG. 1 shows the structure of a multi-layer force ram that can be used in the practice of the invention, in which a porous stopper 2, a silica gel ram, and a silica gel are placed inside a long and thin glass tube 1 having an inner diameter of about 2 to 4 m/m. It is filled with layer 3, magnesium silicate layer 4 and plug 5.

Plugs 2 and 5 are simply for fixing layers 3 and 4;
Glass fiber filter paper strips are conveniently used for this purpose;
Of course, other inert porous materials may be used as long as they can achieve the purpose of fixation (porosity is not required in the plug 5).

The lower layer 3 can be composed of ordinary silica gel, but in practice it is suitable to use silica gel for column chromatography (for example, Wakogel C-100 (trade name), Kieselgel 0.05=0.2 m/m, etc.).

When selecting an adsorbent, criteria include the ability to confirm 10 ppb of contamination, the difficulty in causing tailing, and the short development time. As mentioned above, there is no one that satisfies all of them.

As a result of the test, it was found that acid alumina, which has been frequently used as an adsorbent for column chromatography for a long time, is not very suitable because it has a weak adsorption ability for Af.

Next, layer 4 needs to be selected from an adsorbent that has a stronger adsorption capacity for Af than layer 3.

According to the inventor's knowledge, magnesium silicate gel (for example, Florisil (trade name)) is the most suitable adsorbent for this purpose.

The particle size of the adsorbent is of practical importance as it affects the deployment time.

Generally, particles with a mesh size of 100 mesh or less are good, and particles with a mesh size exceeding 200 mesh are not suitable for practical use due to the time required for development.

Furthermore, the appropriate lengths of layers 3 and 4 are determined by the sample development (extraction) solvent,
Since it varies depending on the type of adsorbent, the inner diameter of the tube 1, etc., it is best to determine it experimentally.

However, in the inventor's experience, when a tube with an inner diameter of 3 m/m was used, it was appropriate to configure layer 3: 15 mm and layer 4: 85 mm, totaling 100 mm.

This value is based on peanuts as a sample, but
This is a standard value that can be applied to other samples as well.

The inner diameter of the tube 1 is preferably 2 to 4 m/m.

The present invention is an application of ascending force ram chromatography, and at the same time attempts to extract Af in a sample using a solvent. Therefore, the solvent used has a strong dissolving power for Af, and at the same time has a strong dissolving power for impurities. weak,
In addition, it is preferable that the adsorbent used has a different migration distance between impurities and Af.

Af is insoluble in typical nonpolar solvents such as hexane and ether, but is well soluble in intermediate solvents between nonpolar and polar, such as chloroform, and other polar solvents, such as acetone, alcohol, or mixtures thereof with water.

However, water-containing solvents generally have low dissolving power for Af, so a large amount of solvent is required, and they are generally difficult to blend with samples and filter, and not only do they take time to develop, but they also tend to cause tailing, making them unsuitable as solvents. be.

Furthermore, since a solvent containing hexane elutes a large amount of oil and fat, a long time is required for development.

On the other hand, chloroform alone does not have a very high ability to extract Af, but when mixed with a small amount of lower aliphatic alcohol, the selective extraction power for Af increases, and this result is better when acetone is used in place of the alcohol. The above mixture is suitable as a solvent for selective extraction of Af.

However, the mixing ratio of chloroform and lower aliphatic alcohol naturally has an appropriate range, and the ratio of the two should be 98.2 to 95.
It was recognized that the best ratio was 5:18 to 4.5 (capacity ratio), especially 97:3*.

Methanol is the best lower aliphatic alcohol, but ethanol, propanol or isopropanol will give almost the same results.

*This solvent ratio is the same as reported by Mr. Holladay mentioned above.

When an Af-containing sample is extracted with the above solvent and the lower end of the force ram is immersed in this extract and left to stand, Af in the sample is mainly adsorbed near the interface between layers 3 and 4.

However, this operation alone causes tailing A in layer 3.
It is difficult to raise f to the interface, resulting in incomplete separation of interfering substances and Af in layer 3, and as a result, the Af concentration at the interface cannot be increased to a discernible value. becomes.

In addition, some types of interfering substances are easily adsorbed at the interface, and they may also produce fluorescence similar to Af, so in some cases it may appear as though Af is present even though Af is not present. There is a risk of misunderstanding.

However, as a result of research, the present inventors have found that by redeploying the column that has undergone the primary deployment with a more polar solvent, it is possible to uniformly raise the Af tailing within the layer 3 to the vicinity of the interface. At the same time, A present in layer 3 or near the interface
It has been discovered that f can be diffused upward into the interior of layer 4.

Therefore, by this operation, Af is concentrated in a narrow band, and at the same time, a substance that interferes with Af detection is diffused (reduced in concentration), and thus the accuracy of Af detection is dramatically improved.

The secondary developing solvent is preferably a solvent that is slightly more polar than the extraction solvent, for example, a solvent prepared by adding a small amount of acetone to the extraction solvent.

What has been experimentally found to be suitable is a mixture of chloroform: lower aliphatic ketone: lower aliphatic alcohol (96:2:2), but even if this is changed to 90:10:2, the results hardly change.

In this secondary development, the action of lower aliphatic ketones is in layer 3.
This is thought to be due to the diffusion of interfering substances located near the boundary region between layer 4 and layer 4 into layer 4, and lower aliphatic alcohol also functions to raise Af tailing within layer 3 to this region. be understood as a thing.

Examples of suitable lower aliphatic ketones include acetone, methyl ethyl ketone, and diethyl ketone.

Examples of lower aliphatic alcohols have been given above.

Note that this alcohol was exemplified above.

Note that this alcohol can be omitted if desired.

The present invention is generally carried out as follows.

That is, 50 g of a test sample such as peanuts was weighed out, and mixed with 100 ml of a chloroform:methanol (97:3) mixture (as mentioned above, methanol can be substituted with ethanol, propanol, etc.) and placed in a high-speed stirring pulverizer ( Grind and stir the sample in a blender for about 1 minute, or grind the sample in a mortar and shake it together with the solvent in a triangular Kolben for about 3 minutes, then suction or centrifugal filtration (static filtration is also acceptable). (requires a long time), the lower end of the millicolumn is immersed in this filtrate.

After about 6 minutes, when the liquid rose to a height of about 7 cm, the ram was taken out, left to dry for 2 minutes in a thermostat at 80°C, and then mixed again with chloroform:acetone:methanol (96:2:2).
Redeployment is performed by immersion in a mixed solution (it has already been mentioned that methanol can be replaced with another lower alcohol and acetone can be replaced with another lower ketone).

After about 4 minutes, when the liquid rose to a height of 7 cm, the column was taken out and dried as before, and the presence or absence of the Af-specific blue-green fluorescent band and, if present, its intensity was observed under long wavelength ultraviolet light of 3650 Å. do.

Since Af always exists near the interface between layers 3 and 4,
Even if similar fluorescent substances are present, they can be identified by the difference in the position of the fluorescent bands.

In this case, if you are skilled, you can make a rough quantification just by observing the fluorescence intensity of the sample with the naked eye, but if you have a known concentration of Af
More accurate quantification is possible by comparison with a standard force ram that has been adsorbed in advance.

The use of a fluorescent densitometer is effective in eliminating visual fatigue errors.

The time required for this analysis is about 25 minutes, which is only about 1/17 of the time required by the official method.

FIG. 2 is a photograph comparing the occurrence of fluorescence in millicolumns prepared from a standard solution containing a certain amount of standard Af B1 solution and a control solution.

From the left in the figure, control, 10ppb, 100ppb, 50
exhibiting fluorescence at 0 ppb and 1000 ppb concentrations;
It is shown that even at a concentration of 10 ppb, it can be clearly distinguished from the control by the development of a clear fluorescent band.

Figure 3 shows a comparison of the fluorescence generation state between a standard Af B1 solution and an uncontaminated peanut extract containing Af B1 at the same concentration.
f B1 10ppb added uncontaminated peanuts, standard Af
B1 100ppb and standard Af B1100ppb
The results of millicolumn analysis of uncontaminated peanuts are shown.

In this case, the interfering substances in the peanut are below layer 3 and layer 4.
As a result, even a dilute concentration of 10 ppb can be detected in the same way as standard Af B1.

Incidentally, the second-order expansion method explained above was used in the millicolumn analysis shown in Figure 2 above.

Figure 4 shows, from the left, an extract obtained by adding Af B1 to a concentration of 10 ppb to uncontaminated peanuts, and an extract of Af-contaminated peanuts (contamination level unknown) after primary and secondary development, and the state of fluorescence in the adsorptive ram. This is a photo showing.

The Af contamination level of the contaminated peanuts on the right is approximately 600pp.
It is estimated to be around b.

As described above, the present invention is a response to the fervent desire for simple and rapid detection and quantification of aflatoxins, and is believed to be a valuable invention in terms of food hygiene.

[Brief explanation of drawings]

FIG. 1 shows an enlarged partially cutaway elevational view of a milli-column used in the practice of the present invention, in which number 1 represents the entire column, numbers 1a and 1b represent the lower and upper ends of 1, respectively, and 2 represents the entire column. 3 shows the silica gel layer, 4 shows the magnenium silicate layer, and 5 shows the upper plug. Figure 2 shows the state of fluorescence generation in a millicolumn of standard aflatoxin solutions at various concentrations (concentrations are clearly indicated in the figure). FIG. 3 shows the fluorescence status in a millicolumn of a standard aflatoxin solution at a concentration of 10 ppb and 100 ppb, respectively, and a peanut extract to which aflatoxin was added at the same concentration. FIG. 4 shows, from the left, the state of fluorescence development after primary and secondary development of a sample in which Af B1 was added to uncontaminated peanuts at a concentration of 10 ppb and a sample of Af-contaminated peanuts with an unknown degree of contamination, respectively.

Claims (1)

[Claims] 1 After immersing and developing the lower part of the adsorption ram, which is made up of a silica gel layer and a magnesium silicate layer stacked in contact with each other from the lower end inside a long and slender glass tube, in a test sample solution extracted with a chloroform/lower alcohol mixture. dry,
A simple method for detecting aflatoxin, characterized in that the dried column is then redeployed with a solvent more polar than the above mixture, dried again, and observed under long wavelength ultraviolet light.