Patent Application
20170160270
This application is based upon and claims the benefit of priority from Japanese Patent Application 2015-236964 filed on Dec. 3, 2015, the entire contents of which are incorporated herein by reference.
Embodiments of the present invention relate to a marker capable of conveniently determining food freshness and a sensing system using the same.
Amines generated from food decomposition (hereinafter, referred to as “biogenic amines” or simply “amines”) are produced through decarbonation of an amino acid and may cause a heath risk such as an allergic disease or food poisoning in fish, shellfish, or meat. Since the biogenic amine is generated even when edible food is processed or stored, the amount of the generated biogenic amines serves as an index indicating freshness of edible food. In recent years, a method of sensing the biogenic amine with high sensitivity and high selectivity is demanded in order to prevent an allergic disease or food poisoning and reduce food wastes.
As a method of sensing a biogenic amine, an instrumental analysis such as gas chromatography or liquid chromatography is known in the art. However, such an instrumental analysis requires a lot of time for pre-treatment performed before measurement, and a device for the instrumental analysis is to be carefully maintained and calibrated at all times. Therefore, the instrumental analysis is expensive.
Meanwhile, a simple and fast method for sensing a biogenic amine using a tetraphenylethene-based fluorescent substance is known in the art. In this sensing technique, the tetraphenylethene-based fluorescent substance is dissolved in a solution. In a first process, the biogenic amine is dissolved in a solution. In a second process, the tetraphenylethene-based fluorescent substance and the biogenic amine are reacted with each other to produce coagulations. It is known that, although a single particle of the tetraphenylethene-based fluorescent substance has a weak fluorescence intensity, coagulations of the tetraphenylethene-based fluorescent substance provide a stronger fluorescence intensity. Therefore, generation of a biogenic amine can be detected based on the increase of the fluorescence intensity. As a result, it is possible to sense deterioration of food freshness.
A lot of types of biogenic amines are generated through food decomposition, and they have different hydrophobicity. For this reason, while a type of the biogenic amine suitable for the solvent employed in the aforementioned sensing method is detected, another type of the biogenic amine having hydrophobicity significantly different from that of this type is not dissolved in the employed solvent and is not detected. Since the type of the biogenic amine is different in each food, it is necessary to change the solvent depending on a type of target food if the aforementioned sensing method of the prior art is employed. This is disadvantageous.
The accompanying drawings, which is incorporated in and constitute a part of this specification, illustrates an embodiment of the invention and together with the description, serve to explain the principles of the invention.
Reference will now be made in detail to the present embodiment of the invention, an example of which is illustrated in the accompanying drawing.
A marker according to this embodiment is a marker for detecting a detection target substance. The marker has an assay reagent having a medium and a composition containing a solvent and fluorescent coagulation factor that has a fluorescence property that changes as it coagulates in presence of the detection target substance. The assay reagent is divided into two or more segments, and each of the assay reagent segments has different hydrophobicity for the solvent.
Embodiments of the present invention will now be described in detail with reference to the accompanying drawings.
According to this embodiment, the assay reagent has a fluorescent coagulation factor that has a fluorescence property that changes as it coagulates in presence of a biogenic amine serving as the detection target substance, a solvent for dissolving the fluorescent coagulation factor, and a medium for retaining the fluorescent coagulation factor and the solvent. The assay reagent is divided into two or more segments, and each of the assay reagent segments has different hydrophobicity for the solvent. Using the marker according to this embodiment, each assay reagent segment has different hydrophobicity for the solvent. Therefore, it is possible to detect many types of biogenic amines with high accuracy.
In general, if food is kept aside, a change occurs in odor, appearance, texture, taste, and the like as time elapses, and finally, the food becomes inedible. Such a decay of food is called deterioration, putrefaction, or spoilage, and is also typically described as “food is rotten.” The food deterioration occurs due to microorganisms, insects, autolysis, chemical reasons (oxidation of lipide or browning), or physical reasons (such as injury, crush, or damage). In many cases, food becomes inedible due to spoilage caused by proliferation of microorganisms (putrefactive bacteria), and this is called “decomposition” in a broad sense.
A process of generating harmful products or an unpleasant odor when food is decomposed due to a microorganism reaction generated in proteins of food is called “putrefaction.” In contrast, an inedible food state in which carbohydrate or fat and oil is decomposed due to a microorganism reaction, and a flavor of food is degraded may be distinguished from deterioration or spoilage in some cases. In addition, an amine component called various biogenic amines such as ammonia or trimethylamine is a main factor of a putrefactive odor.
For this reason, in order to analyze a decomposition level of food having rich protein such as meat or fish, it is useful to quantify this biogenic amine component. As a biogenic amine quantification analysis method, fast liquid chromatography is generally employed. However, a lot of time and cost are necessary in a complicated sample pretreatment, determination of a measurement time, and the like.
A nitrogen compound of food predominantly contains protein, and protein is hydrolyzed by a microorganism enzyme or a food enzyme to produce polypeptide, simple peptide, or an amino acid. In addition, the amino acid is decomposed through a deamination, transamination, or decarbonation reaction to produce a biogenic amine.
The biogenic amine produced from the amino acid includes, for example, 1,2-ethylenediamine, 1,3-propanediamine, 1,4-butanediamine, 1,5-pentanediamine, 1,6-hexanediamine, spermidine, spermine, histamine, tryptamine, or the like.
A fluorescent coagulation factor according to this embodiment refers to a fluorescent substance that coagulates or flocculates in presence of an amine to generate a change in a distribution or intensity of a fluorescence spectrum or an excitation spectrum or a change in a fluorescence property such as a fluorescence lifetime. As such a fluorescent coagulation factor, for example, coagulation-induced luminescent molecules discussed in JP 2012-51816 A may be employed. The coagulation-induced luminescent molecules do not emit fluorescent light even by irradiating excitation light while it is dissolved in a solvent. However, when they coagulate, fluorescent light is emitted. For example, the coagulation-induced luminescent molecules may be a tetra(aryl)ethene derivative as expressed in the following General Formula (I).
In this formula, the elements R1, R2, R3, and R4 are independent from each other and are selected from a group consisting of —COOM1, —(CH2)m—COOM2, —X—(CH2)n—COOM3, —Y—(CH2)o—Z—(CH2)p—COOM4 (where “M1,” “M2,” “M3,” and “M4” are independent from each other and denote a hydrogen atom or a cation, “X,” “Y,” and “Z” are independent from each other and denote “—O—,” “—NH—,” or “—S—,” and “m,” “n,” “o,” and “p” are independent from each other and denote integers between 1 to 6), a hydrogen atom, a halogen atom, a hydroxyl group, a nitro group, a carbamoyl group, an alkyl group having a carbon number of 1 to 6, a haloalkyl group having a carbon number of 1 to 6, an alkenyl group having a carbon number of 2 to 6, a cycloalkyl group having a carbon number of 3 to 10, an alkyloxy group having a carbon number of 1 to 6, an acyl group having a carbon number of 2 to 6, an amino group, an alkylamino group having a carbon number of 1 to 6, an aryl group having a carbon number of 6 to 10, and a heteroaryl group having a carbon number of 5 to 10. Meanwhile, at least two of the elements R1, R2, R3, and R4 are independent from each other and are selected from a group consisting of —COOM1, —(CH2)m—COOM2, —X—(CH2)n—COOM3, and —Y—(CH2)o—Z—(CH2)p—COOM4 (where “M1,” “M2,” “M3,” and “M4” are independent from each other and denote a hydrogen atom or a cation, “X,” “Y,” and “Z” are independent from each other and denote “—O—,” “—NH—,” or “—S—,” and “m,” “n,” “o,” and “p” are independent from each other and denote integers between 1 to 6).
Note that the cation of the Chemical Formula (1) described above may be either an organic or inorganic cation without a particular limitation. For example, the cation may include, for example, ammonium, alkali metal, alkali earth metal, pyridinium, or the like. In addition, if two or more cations exist in a single molecule, different cations may be included.
In the aforementioned General Formula (I), the tetra(aryl)ethene derivative coagulates because its solubility is degraded in a solution due to a hydrogen bond or an electrostatic interaction (hereinafter, also referred to as a “reaction”) between a carboxyl group and an amine in a molecule. If excitation light such as ultraviolet rays is irradiated onto this coagulating tetra(aryl)ethene derivative, it emits fluorescent light. According to this embodiment, a composition including the solvent and the fluorescent coagulation factor retained in a medium is created such that it has a concentration at which an unreacted fluorescent coagulation factor does not coagulate or precipitate, that is, no saturation occurs.
According to this embodiment, a solvent capable of dissolving the fluorescent coagulation factor is employed, and a difference of the hydrophobicity parameter LogP of all or a part of the biogenic amines as a detection target is set to “1.53” or smaller. If the difference of the hydrophobicity parameter LogP between the solvent and the biogenic amine as a detection target is greater than “1.53,” mutual solubility between the biogenic amine and the solvent is degraded, and the resulting coagulation becomes uneven. As a result, sensitivity of the mark may be degraded.
Note that the hydrophobicity parameter LogP refers to a distribution coefficient of a substance in water and 1-octanol and is calculated using a software program “Molecular Modeling Pro. Plus Version 7.0.4” produced by Norgwyn Montgomery Software, Inc.
According to this embodiment, since the marker is used to detect a time course of freshness deterioration, the solvent is preferably seldom volatilized and dried out under the atmosphere for a certain period of time. In addition, according to this embodiment, since the marker is added or neighbored to food, the solvent preferably has high safety for a human body. Furthermore, according to this embodiment, the solvent may be a solvent mixture obtained by mixing two or more types of solvents.
A glycol-based solvent having a high boiling point and low toxicity is preferably employed as the solvent according to this embodiment. Such a glycol-based solvent may include, for example, an ethylene glycol-based solvent such as polyethylene glycol monomethyl ether, diethylene glycol ethyl methyl ether, polyethylene glycol dimethyl ether, triethylene glycol butyl methyl ether, and diethylene glycol butyl methyl ether, or a propylene glycol-based solvent such as propylene glycol monomethyl ether, propylene glycol monobutyl ether, and propylene glycol dimethyl ether.
As illustrated in
As the difference of the hydrophobicity parameter LogP between the solvent and the biogenic amine decreases, solubility of the biogenic amine increases. As the difference of the hydrophobicity parameter LogP between the solvent and the biogenic amine increases, solubility of the biogenic amine decreases. That is, mutual solubility is high between substances having the smaller difference of the hydrophobicity parameter LogP. If the difference of the hydrophobicity parameter LogP between the solvent and the detection target substance (decomposition substance) is small, this means that the solvent and the detection target substance are easily evenly mixed with each other.
Therefore, in the case of the glycol-based solvent illustrated in
(1) an assay reagent having a total carbon number of the terminal substituent group set to “2” in the case of a monoethylene glycol-based solvent or a diethylene glycol-based solvent, having a total carbon number of the terminal substituent group set to “2 to 3” in the case of a triethylene glycol-based solvent, or having a total carbon number of the terminal substituent group set to “2 to 5” in the case of a tetraethylene glycol-based solvent;
(2) an assay reagent having a total carbon number of the terminal substituent group set to “3 to 4” in the case of a monoethylene glycol-based solvent, having a total carbon number of the terminal substituent group set to “3 to 6” in the case of a diethylene glycol-based solvent, having a total carbon number of the terminal substituent group set to “3 to 6” in the case of a triethylene glycol-based solvent, or having a total carbon number of the terminal substituent group set to “3 to 7” in the case of a tetraethylene glycol-based solvent; and
(3) an assay reagent having a total carbon number of the terminal substituent group set to “5 to 8” in the case of a monoethylene glycol-based solvent, having a total carbon number of the terminal substituent group set to “6 to 8” in the case of a diethylene glycol-based solvent, having a total carbon number of the terminal substituent group set to “7 to 8” in the case of a triethylene glycol-based solvent, or having a total carbon number of the terminal substituent group set to “7 to 8” in the case of a tetraethylene glycol-based solvent.
According to this embodiment, any medium may be employed without a particular limitation as long as it can retain a composition (mixed solution) containing the fluorescent coagulation factor and the solvent described above. Considering retention of the composition (mixed solution), the medium preferably has a certain or higher level of porosity. For example, a porous substrate, a mesh structure, or the like may be employed.
Such a medium may include, for example, cellulose fiber, paper, fabric, a filter, a sponge, or the like. In particular, a membrane filter formed of a cellulose acetate material is preferably employed because it can increase the fluorescence intensity.
The medium is preferably selected from materials having a refractive index similar to that of the solvent according to this embodiment. If the employed medium has a refractive index similar to that of the solvent, the fluorescent light generated from the inside of the medium is not hindered. Therefore, it is possible to obtain a stronger fluorescence intensity and easily determine freshness.
A substrate capable of supporting the assay reagent may also be employed as necessary. The substrate preferably has a solvent resistance against the solvent used to dissolve the fluorescent coagulation factor. In addition, the substrate is preferably prohibited to emit fluorescent light by itself. Any material may be employed in the substrate as long as it does not have a wavelength similar to a fluorescence wavelength that the fluorescent coagulation factor emits.
Such a member may include, for example, a Teflon (registered trademark) sheet, a polyimide sheet, a polyester film, a polyacetal sheet, a nylon sheet, a polycarbonate sheet, a polypropylene sheet, a polyethylene sheet, a PET film, a plastic sheet such as a vinyl chloride sheet, a glass plate, or the like.
The marker according to this embodiment detects a food freshness state by detecting a change of the fluorescence property caused by a reaction between the amine and the fluorescent coagulation factor retained on the medium as described above. Fluorescent light emitted by irradiating ultraviolet light onto the marker according to this embodiment from an UV light source unit is recognized using a light emission detection unit to determine a food freshness state.
A sensing system according to this embodiment includes the marker according to this embodiment, an ultraviolet (UV) light source unit that emits ultraviolet light onto the marker, and a light emission detection unit that detects an image pattern exhibited on the marker by irradiating ultraviolet rays. The detection target substance is detected on the basis of the image pattern exhibited on the marker by irradiating ultraviolet rays to determine a food freshness state. Here, the light emission detection unit refers to a visual inspection or an imaging device such as digital camera.
According to this embodiment, if the food freshness state is determined through a visual inspection as the light emission detection unit, the inspection is preferably performed in a dark place by avoiding a visible light atmosphere. In addition, using a fluorophotometer, it is possible to determine the freshness state with higher accuracy. Furthermore, by recognizing an image (image pattern) obtained using a charge-coupled device (CCD) image sensor or a complementary metal oxide semiconductor (CMOS) image sensor such as a digital camera, it is possible to perform the determination with higher accuracy.
Such an image electronically processed using a digital camera and the like can convert a weak fluorescent image into an image having a higher contrast. This method would be more useful when a user wants to determine a slight fluorescence intensity difference, that is, when a user wants to determine a very small difference of the amount of the biogenic amine. Furthermore, if a colorimetric function through image processing is provided in a camera-installed smart phone or the like, it is possible to determine freshness with an automatic determination function.
Examples and comparative examples of the present invention will now be described in more detail. Note that the following examples are not intended to limit the present invention.
As the fluorescent coagulation factor, a compound (1) having a carboxyl group in positions of the elements R1 and R3 and a hydrogen atom in positions of the elements R2 and R4 in the general formula (I) is employed. The compound (1) is dissolved in the following solvent by 0.02 weight % to produce the fluorescent liquids A and B.
First, as illustrated in
Then, as illustrated in
As illustrated in
The fluorescent state was observed by picking out the marker samples from the glass container G and irradiating ultraviolet rays under an appropriate dark room state. Their fluorescent states were captured using a digital camera. The irradiation of ultraviolet rays was performed using a compact black light source.
From the images of
It is recognized that a change of the fluorescence intensity of the marker sample B is insignificant relative to the marker sample A of the fluorescent liquid A in both the glass container with a raw food material P (fish paste) and the empty glass container. In addition, even in the fluorescent state after three days, there is no significant increase of the fluorescence intensity.
That is, a high sensitive marker for recognizing a change of the freshness state in the food material (fish paste) used in this test is the marker sample A of the fluorescent liquid A in which polyethylene glycol dimethyl ether is employed as a solvent.
As a result of the evaluation described above, it is recognized that it is necessary to create a fluorescent liquid suitable for the corresponding food material in order to prepare a marker suitable for various food materials.
Dependence on the solvent in the fluorescence spectrum was evaluated for the compound (1) by selecting spermidine of a biogenic amine as a detection target substance. Various parameters of the used solvent and the spermidine are shown in Table 1. The hydrophobicity parameter LogP was calculated using Molecular Modeling Pro. Plus Version 7.0.4, produced by Norgwyn Montgomery Software, Inc. First, the structure was optimized using a molecular dynamics calculation (MM2), and the hydrophobicity parameter LogP was calculated using a three-dimensional structure activity relationship (three-D QSAR terms).
Fluorescence spectra obtained by adding an eight molar equivalent (500 μmol) of spermidine to each solvent of the compound (1) are illustrated in
From the fluorescence spectra illustrated in
As a comparative example in which spermidine is used as a detection target substance, a fluorescence spectrum of the compound (1) obtained by using dibutyl maleate having a hydrophobicity parameter LogP of 2.13 as the solvent (ΔLogP=1.91) was evaluated. As a result, the fluorescence intensity was remarkably reduced by adding spermidine and irradiating black light for fourteen hours.
However, there was no remarkable reduction in the fluorescence intensity when phenethylamine (LogP=1.84, ΔLogP with dibutyl maleate<0.29) having a hydrophobicity parameter LogP greater than that of the spermidine is used as a detection target substance.
From the evaluation result described above, if a difference of the hydrophobicity parameter LogP between the solvent used in the assay reagent of the marker and the detection target substance is greater than 1.53, the detection target substance is not dispersed in a molecular state in the solvent, but forms a minute droplet. As a result, it is conceived that the coagulation formed from the detection target substance and the fluorescent coagulation factor becomes uneven, and the retention of the fluorescence intensity is degraded.
In contrast, if the difference of the hydrophobicity parameter LogP is equal to or smaller than 1.53, an even coagulation network between the fluorescent coagulation factor and the detection target substance is easily formed, and such a coagulation has excellent durability. Meanwhile, if a solvent has a terminal “—OH” group as in the solvent of No. 4, it forms a hydrogen bond with a “—COOH” group of the fluorescent substance. Therefore, it is conceived that an interaction with a decomposed substance is limited. Such a solvent is advantageously added to a part of a solvent mixture in order to adjust sensitivity of the marker (necessary to adjust sensitivity depending on food). However, if the entire solvent is the No. 4 type, a coagulation rate is reduced.
A fluorescence lifetime was evaluated immediately after solely adding the compound (1) to the solvents No. 1 to 4, immediately after adding spermidine, after irradiating black light for ten hours, and after irradiating black light for fourteen hours. In addition, as a durability test, a fluorescence lifetime was evaluated after the sample is kept aside for thirteen days under the room light at the room temperature while it is opened to the atmosphere. The result of evaluation is shown in Table 2. Note that the fluorescence lifetime of Table 2 is obtained by approximating the attenuation function of the fluorescence into two types of exponential functions, and the numbers in the parenthesis refer to χ2 coefficients.
Table 2 shows a change of the fluorescence lifetime of the compound (1) by adding spermidine as a detection target substance (decomposition substance) and a change of the fluorescence lifetime as a result of the durability test.
As shown in Table 2, in the cases of Nos. 1, 2, and 3 in which the fluorescence intensity increases by adding spermidine, the main fluorescence lifetime is reduced by adding spermidine. Meanwhile, in the case of No. 4 in which the fluorescence intensity does not increase, the main fluorescence lifetime is not reduced. This is because an interaction between the compound (1) and the spermidine increases, that is, because a coagulation is generated.
Although the fluorescence intensity is reduced by irradiating UV light, the fluorescence lifetime increases accordingly. It is conceived that this is because an interaction between the compound (1) and the spermidine is weakened. Meanwhile, the rightmost fluorescence lifetime in which the fluorescence intensity is not reduced even through the durability test does not increase. As a result, it is possible to know that this fluorescence lifetime can be used in freshness determination.
While certain embodiments have been described, these embodiments have been presented by way of example only, and are not intended to limit the scope of the inventions. Indeed, the novel embodiments described herein may be embodied in a variety of other forms; furthermore, various omissions, substitutions and changes in the form of the embodiments described herein may be made without departing from the spirit of the inventions the accompanying claims and their equivalents are intended to cover such forms or modifications as would fall within the scope and spirit of the inventions.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2015-236964 | Dec 2015 | JP | national |
