JP5806004B2 - Sweetness test method and sensor film for sweetness test - Google Patents

Description

  The present invention relates to a technique for inspecting a tastant based on a change in potential of a sensor film with respect to a tastant, and more particularly to a technique for performing a test for sweetness with high sensitivity and selectivity.

  As a technique for inspecting a tastant, a method is known that utilizes a membrane sensor containing lipid to change its membrane potential in response to a tastant.

  The membrane sensor is a membrane formed by mixing a polymer material such as PVC, which is the base of the sensor, a plasticizer, and a lipid that exhibits a response to a taste substance at a predetermined ratio, and has a membrane potential. Can be output via the electrode, and by selecting the material of the lipid or plasticizer, the mixing ratio, etc., it is possible to construct a membrane sensor having a different response to taste substances.

  Taste substances are basically classified into those showing salty taste, sour taste, bitter taste, umami taste, and sweet taste. However, in the examination using a membrane sensor so far, the sensitivity and selection of sweet taste substances compared to other basic taste substances. However, the improvement in sensitivity and selectivity for this sweet substance is desired.

  As one solution to this problem, the applicant of the present invention modified the surface of a membrane main body made of a polymer material, a plasticizer, and a lipid with a polyhydric phenol such as polyhydric phenolic acid or hydrolyzable tannic acid, The following Patent Document 1 proposes a method for inspecting a sweet substance based on the finding that sensitivity and selectivity to a substance can be obtained.

JP 2007-217630 A

  However, in the above-mentioned Patent Document 1, it cannot be said that the reasoning that sensitivity and selectivity for sweet substances can be obtained by surface modification of the membrane main body with polyhydric phenol, the conditions for using the invention are Limited and insufficient in terms of applicability.

  The present invention logically derives the structural principle for improving the sensitivity and selectivity for sweet substances based on various experiments, and provides a sweetness test method and a sweetness test membrane sensor that are widely applicable to the principle. It is aimed.

In order to achieve the above object, the sweetness test method of the present invention comprises:
A membrane sensor comprising a polymer material, a plasticizer, and a weakly acidic substance having a carboxyl group or a phosphate group and having a hydrophobic portion is immersed in an alkaline pretreatment liquid containing a metal cation. Adsorbing the metal cations on the membrane surface by interaction with the weakly acidic substance of the membrane sensor;
The membrane sensor to which the metal cation is adsorbed is immersed in a liquid to be measured containing a sugar substance, and the metal cation is separated from the membrane surface by interaction with the sugar substance, thereby changing the membrane potential in the negative direction. It is characterized by including a stage.

Moreover, the film | membrane sensor for sweetness inspection of Claim 2 of this invention is the following.
A polymer material, a plasticizer, and a weakly acidic substance having a carboxyl group or a phosphate group and having a hydrophobic portion are mixed to form a film , and a metal cation interacts with the weakly acidic substance. It is characterized in that it is adsorbed on the film surface by action .

A sweetness test membrane sensor according to claim 3 of the present invention is the sweet taste test membrane sensor according to claim 2,
It is characterized by being contained in a ratio of 800 mg of polymer material PVC, 1 ml of plasticizer DOPP, and 30 to 120 mg of weakly acidic substance trimellitic acid .

The sweetness test membrane sensor according to claim 4 of the present invention is the sweet taste test membrane sensor according to claim 3,
Lipid TDAB is contained at a ratio of 1 mg or less .

  The sweetness test method of the present invention comprises a membrane sensor formed by mixing a polymer material, a plasticizer, and a weakly acidic substance having a carboxyl group or a phosphate group and having a hydrophobic portion, and an alkaline substance containing a metal cation. In the pretreatment liquid, the metal cation is adsorbed on the surface of the film by the interaction with the weakly acidic substance of the membrane sensor. Since the cations are separated from the membrane surface by the interaction with the sugar substance and the membrane potential is changed in the negative direction, sweetness can be detected with higher sensitivity and higher selectivity than in the past.

The sweetness test film sensor of the present invention is formed into a film by mixing a polymer material, a plasticizer, and a weakly acidic substance having a carboxyl group or a phosphate group and having a hydrophobic portion , Further, since metal cations are adsorbed on the membrane surface, the membrane sensor is immersed in a liquid to be measured containing a sugar substance, and the metal cations are separated from the membrane surface by interaction with the sugar substance, and the membrane in the Turkey changing the potential in the negative direction, it can be performed sweetness detection with and higher selectivity to higher sensitivity conventionally.

The figure for demonstrating the sweetness detection principle of this invention Structure example and system diagram of membrane sensor for sweetness detection The figure which shows the kind of weakly acidic substance (sweet responsive substance) to be contained in the membrane sensor, the content and the measurement result The figure which shows the difference in the response to the content ratio with lipid when trimellitic acid is used as a weakly acidic substance (sweet responsive substance) A diagram showing the measurement results of FIG. 4 in a three-dimensional graph The figure which shows the difference in the response of the conventional sensor and the example sensor to basic taste The figure showing the correlation of the response of the film | membrane sensor of embodiment with respect to the sugar solution of known sweetness degree, and sweetness degree Measurement results for sucrose solution Measurement results for glucose solution Measurement results for fructose solution Measurement results for raffinose solution Measurement results for isomaltoligosaccharide solution Measurement results for artificial sweetener solution Measurement results for sucrose solution when lipid is changed to TOMA Figure showing the relationship between the number of sugar hydroxyl groups and the response potential Figure showing the relationship between the number of sugar hydroxyl groups and the response potential Diagram showing the relationship between the components of the pretreatment liquid and the response potential Diagram showing the relationship of response potential depending on the presence or absence of chloride in the pretreatment liquid The figure which shows the difference in the response to the content ratio with lipids when using weakly acidic substances (sweet responsive substances) other than trimellitic acid The figure which shows the difference in the response to the content ratio with lipids when using weakly acidic substances (sweet responsive substances) other than trimellitic acid The figure which shows the difference in the response to the content ratio with lipids when using weakly acidic substances (sweet responsive substances) other than trimellitic acid The figure which shows the difference in the response to the content ratio with lipids when using weakly acidic substances (sweet responsive substances) other than trimellitic acid The figure which shows the difference in the response to the content ratio with lipids when using weakly acidic substances (sweet responsive substances) other than trimellitic acid The figure which shows the difference in the response to the content ratio with lipids when using weakly acidic substances (sweet responsive substances) other than trimellitic acid The figure which shows the difference in the response to the content ratio with lipids when using weakly acidic substances (sweet responsive substances) other than trimellitic acid The figure which shows the difference in the response to the content ratio with lipids when using weakly acidic substances (sweet responsive substances) other than trimellitic acid The figure which shows the difference in the response to the content ratio with lipids when using weakly acidic substances (sweet responsive substances) other than trimellitic acid The figure which shows the difference in the response to the content ratio with lipids when using weakly acidic substances (sweet responsive substances) other than trimellitic acid The figure which shows the difference in the response to the content ratio with lipids when using weakly acidic substances (sweet responsive substances) other than trimellitic acid The figure which shows the difference in the response to the content ratio with lipids when using weakly acidic substances (sweet responsive substances) other than trimellitic acid The figure which shows the difference in the response to the content ratio with lipids when using weakly acidic substances (sweet responsive substances) other than trimellitic acid The figure which shows the difference in the response to the content ratio with lipids when using weakly acidic substances (sweet responsive substances) other than trimellitic acid The figure which shows the difference in the response to the content ratio with lipids when using weakly acidic substances (sweet responsive substances) other than trimellitic acid The figure which shows the difference in the response to the content ratio with lipids when using weakly acidic substances (sweet responsive substances) other than trimellitic acid The figure which shows the difference in the response to the content ratio with lipids when using weakly acidic substances (sweet responsive substances) other than trimellitic acid

First, the principle of the sweetness test method of the present invention will be described.
The inventor of the present application is not only a polymer material (PVC), a plasticizer (DOPP), a lipid (TDBA) (including a case where no lipid is included) constituting basic components of a conventional taste sensor film sensor. By using a membrane sensor formed by mixing a weakly acidic substance having a carboxyl group or a phosphate group, and immersing it in an alkaline pretreatment liquid having a metal cation at the pretreatment stage, It has been found that a sweetness test can be performed with higher sensitivity and selectivity than when the membrane sensor disclosed in Patent Document 1 is used.

  Although the experimental data will be described later, the basis for the ability to selectively detect sweetness with high sensitivity has become clear from the experimental data. This will be described below.

  FIG. 1 schematically shows the principle of the sweetness test method of the present invention. First, a polymer material such as PVC, a plasticizer such as DOPP, a carboxyl group (or trimellitic acid, etc.) A membrane sensor is prepared in which a weakly acidic substance having a phosphate group and a hydrophobic portion is mixed at a predetermined mixing ratio.

Since the lipid itself, as will be described later hardly involved in the response to sweetness, which is a main sweetness responsive material weakly acidic material (in FIG. 1 R-COO ^- hereinafter) will be described.

When a membrane sensor containing a weakly acidic substance is immersed in an alkaline pretreatment liquid containing a metal cation such as K (potassium) or Na (sodium) for several minutes, the metal cation (K ⁺ ) and the weakly acidic substance of the membrane sensor As a result of this interaction, metal cations (K ⁺ ) are adsorbed on the membrane surface (a).

  Then, the membrane sensor to which the metal cation has been adsorbed is lightly washed, and the excess pretreatment liquid is removed, and the membrane sensor is immersed in a liquid to be measured containing a sugar substance (b).

  At this time, due to the interaction between the metal cation adsorbed on the membrane surface and the hydroxyl group of the sugar substance, the metal cation is separated from the membrane surface, and the potential of the membrane sensor is changed in the negative direction (c).

  The reason why the above principle is valid is that, as will be described later, high responsiveness to sweetness can be obtained even if the material and content ratio of weakly acidic substances contained in the membrane sensor are variously changed. As for chloride, iodide and bromide, there are changes in response due to differences in metal cations, but it is clear from the fact that the change in response can hardly be confirmed even if the type of anion side (chlorine, iodine, bromine) changes. is there. Hereinafter, the experimental result will be described.

  First, regarding the composition of the membrane sensor, a weakly acidic substance (sweet responsive substance) and lipid TDAB (tetradodecyl ammonium bromide) shown in FIG. A conventional method, that is, using 10 ml of THF (tetrahydrofuran) as a solvent, is dissolved in a film on a glass plate, and the solvent is volatilized to form a film body having a thickness of 200 μm.

  FIG. 2 shows an example of the structure of the actual membrane sensor 100 and the reference electrode 200 and a measurement system. The membrane sensor 100 covers a hole provided on the side surface of an insulating cylinder 101 such as glass. A membrane body 102 is fixed to the outer surface, a buffer layer 103 in which a mixed solution of electrolyte KCl and AgCl is solidified with agar, and an electrode 104 made of a conductor Ag / AgCl are provided inside the cylinder 101. A part of the back surface is in contact with the buffer layer 103 so that the potential of the film body 102 can be output to the outside through the buffer layer 103 and the electrode 104. As shown in FIGS. 7 and 8 of Patent Document 1, the structure of the film sensor may be a structure in which the film main body overlaps the electrode on the substrate in addition to the cylindrical type.

  The reference electrode 200 is for outputting a reference value Va of the potential of the membrane sensor 100. The membrane sensor 100 and the reference electrode 200 are immersed in a container 300 containing a liquid to be measured at regular intervals. The output Va of the reference electrode 200 is supplied to the voltage detection circuit 301 together with the output Vb of the membrane sensor 100, and the voltage Vb−Va is obtained as the response voltage Vx of the membrane sensor 100 with respect to the sample liquid. Is converted into a computer and given to an arithmetic processing unit 303 formed of a computer to perform voltage arithmetic processing, storage processing, and the like. The actual measurement result is the difference (relative value) between the measured value of the liquid to be measured and the measured value of the reference liquid.

  The reference electrode 200 has a structure in which the cylindrical body 101 and the membrane body 102 are removed from the membrane sensor 100, that is, a buffer layer 203 in which a mixed solution of electrolytes KCl and AgCl is hardened with agar, and an electrode 204 made of a conductor Ag / AgCl. The surface potential of the buffer layer 203 is output via the electrode 204.

  The measurement results in FIG. 3 use the membrane sensor having the above structure and a reference electrode, make the solution to be measured a 300 mM concentration and a 1M sucrose solution, and use an alkaline pretreatment solution containing a metal cation as 100 mM KCl, 10 mM KOH. , A mixed solution of 30% ethanol with a pH of 12.5, and before immersing in this pretreatment solution, immerse in a reference solution (1 mM KCl), and the measured value Vx1 at that time and when immersing in the solution to be measured The difference Vx2-Vr1 from the measured value Vx2 is used as the response potential of the membrane sensor for the solution to be measured. The solution to be measured contains 30 mM KCl as a supporting electrolyte and 0.3 mM tartaric acid for pH adjustment, and the pH is adjusted to about 3.4.

  No. 3 in FIG. The sweetness-responsive substances 2 to 5 are weakly acidic substances having a carboxyl group and having a hydrophobic part, but are classified into polyhydric phenols used when modifying the membrane body part before measurement in Patent Document 1. It is what is done. No. No. 1 trimellitic acid, No. 1 Each of the sweet responsive substances 6 to 29 is a non-polyhydric phenol, a weakly acidic substance having a carboxyl group and having a hydrophobic part.

  No. The sweet responsive substances 30, 31 are weakly acidic substances having a phosphate group and having a hydrophobic part.

  As is apparent from the results of measurement using the above method with the membrane sensor mixed with the sweet taste responsive substance shown in FIG. 3, the response voltage (mV) of the 30 mM sucrose solution and the 1 M sucrose solution. It can be seen that the absolute value of is correlated correctly with the magnitude of the concentration.

  From this result, a highly sensitive measurement is performed by performing a sweetness test by the above method using a membrane sensor containing at least a non-polyhydric phenol, a carboxyl group or a phosphate group, and a weakly acidic substance having a hydrophobic portion. Can be said.

  No. In the case of a membrane sensor containing a weakly acidic substance of 2 to 5 polyhydric phenols, a membrane sensor whose surface is modified and a membrane sensor that is integrally manufactured by mixing these weakly acidic substances are detected with sweetness. Since the identity of the principle is not proved, it has novelty and inventive step.

  Next, more detailed characteristics when trimellitic acid is used as the weakly acidic substance (sweet responsive substance) will be described.

  FIG. 4 and FIG. 5 show the measurement results of the response potential with respect to the sucrose solution when the contents of trimellitic acid and lipid TDAB are changed. FIG. 4 shows the measurement results numerically, and FIG. 5 shows the results as a three-dimensional graph.

  From this result, it can be seen that a high response potential is obtained in a region where the lipid TDAB content is low (including zero), and that a high response voltage is obtained at around 100 mg for trimellitic acid.

  Moreover, although the practical sensitivity is acquired in the wide range shown in FIG. 4, FIG. 5, in this range, especially lipid TDAB 1.0mg or less (including 0), trimellitic acid 80-120mg. The responsiveness of the film sensor contained in the range is high, and more sensitive inspection can be performed.

  Next, the result of having measured the response with respect to each taste of the film | membrane sensor containing 100 mg of trimellitic acid and 1 mg of lipid TDAB among the said range is shown in the graph of FIG. In FIG. 6, “GL1” is a characteristic of the membrane sensor of the above embodiment containing trimellitic acid 100 mg and lipid TDAB 1 mg, and “GL0” is a surface modification of the membrane body with gallic acid disclosed in Patent Document 1. This is a characteristic of the film sensor.

  As is apparent from the measurement results of FIG. 6, the membrane sensor “GL1” of the embodiment has a significantly higher response potential to the thin sucrose solution than the conventional membrane sensor “GL0”, and is difficult to distinguish from the conventional sweet taste. Sensitivity to umami is very small.

  Although the response potential is higher for salty and sour taste than the conventional “GL0” membrane sensor, it is more difficult to distinguish between salty and sour because each can be inspected by a highly selective membrane sensor. It is a great merit that the selectivity to nasty taste is high.

  FIG. 7 shows a comparison (a) of the response values of the membrane sensor “GL1” of the above-described embodiment with respect to various sugar substance solutions of known sweetness, and a graph (b) in which the correlation is examined.

  FIG. 7 shows that the membrane sensor of this embodiment has a high correlation with the known sweetness level.

  8 to 12 show the responses of the membrane sensor of the above embodiment to the concentrations of different types of sugar solutions. FIG. 8 shows sucrose, FIG. 9 shows glucose (glucose), and FIG. Is a sample solution in which raffinose (oligosaccharide) is dissolved in pure water or a reference solution (30 mM KCl + 0.3 mM tartaric acid pH 3.5). Weight percent).

  From this measurement result, it can be seen that the membrane sensor of the embodiment can obtain a response voltage corresponding to the concentration of any sugar solution.

  FIG. 13 shows that the concentration of a sample solution obtained by dissolving acesulfame potassium (sweetness level 200) and sodium saccharin (sweetness level 500), which are artificial sweeteners, in the reference solution is the same sweetness as a 10% sucrose and 30% sucrose solution. Thus, the measurement result is shown, and a measurement result almost equal to that of the sucrose solution is obtained. Therefore, it can be seen that the membrane sensor of the embodiment can also detect sweetness for the artificial sweetener.

  Note that the material of the lipid included in the membrane sensor is not limited to the TDAB. FIG. 14 shows the results of measurement using TOMA (trioctylmethylammonium chloride), which has been conventionally known as a lipid used in membrane sensors.

  From this measurement result, it can be seen that a sufficiently high response potential can be obtained even when TOMA is used as the lipid contained in the membrane sensor.

Next, measurement results for verifying the validity of the above-described measurement principle will be shown.
First of all, according to the above principle, the response potential is obtained by the action of the metal cation adsorbed on the membrane sensor and the hydroxyl group of the sugar of the liquid to be measured, so the sensitivity should change depending on the number of hydroxyl groups of the sugar. .

FIG. 15 and FIG. 16 are measurement results that clearly show this, and FIG. 15 shows the following sugar substances having 1 to 6 hydroxyl groups,
Number 1 Ethanol (Hydroxyl group number 1)
Number 2 Ethylene glycol (2 hydroxyl groups)
Number 3 Glycerol (3 hydroxyl groups)
Number 4 Erythritol (sugar alcohol) (number of hydroxyl groups 4)
No. 5 Xylitol (sugar alcohol) (5 hydroxyl groups)
No. 6 Sorbitol (sugar alcohol) (6 hydroxyl groups)
It is the result of having measured the 30 mM solution of this with the film | membrane sensor of embodiment.

In addition, FIG. 16 shows the following sugar substances having hydroxyl groups of 5, 8, and 11;
Number 1 Fructose (Monosaccharide, hydroxyl number 5)
Number 2 Glucose (Monosaccharide, Hydroxyl group number 5)
No. 3 Lactose (disaccharide, hydroxyl number 8)
Number 4 Maltose (disaccharide, hydroxyl number 8)
No. 5 Sucrose (disaccharide, hydroxyl number 8)
Number 6 Trehalose (disaccharide, hydroxyl number 8)
No. 7 Raffinose (trisaccharide, hydroxyl number 11)
It is the result of having measured the 30 mM solution of this with the film | membrane sensor of embodiment.

  As is clear from these figures, it can be clearly seen that the response potential increases in accordance with the number of hydroxyl groups of the sugar substance. Although not shown, the same measurement results are obtained for sugar substances such as 2-deoxyglucose, xylose, galactose, 2-deoxygalactose, mannose, and lyxose.

  Next, with regard to the alkaline pretreatment liquid, in the above example, a mixed liquid having a pH of 12.5 consisting of 10 mM KOH, 100 mM KCl, and 30% ethanol was used, but practical sensitivity can be obtained with other components. I have confirmed.

FIG. 17 shows the components of the pretreatment liquid and the measurement results. Number 1 is the combination of 10 mM KOH and 100 mM KCl, number 2 is the combination of 10 mM LiOH and 100 mM LiCl, and number 3 is Combination of 10 mM NaOH and 100 mM NaCl, number 4 is a combination of 10 mM Ca (OH) ₂ and 100 mM CaCl ₂ , and number 5 is a combination of 1% TMAH (tetramethylammonium hydroxide) and 100 mM KCl .

  As is apparent from the measurement results, a response to sweetness can be obtained by using an alkaline pretreatment liquid containing a metal cation. In particular, in addition to the above number 1, a combination of number 3 NaOH and NaCl, A combination of 5 TMAH and KCl has a high sensitivity.

As is apparent from the above detection principle, even if a liquid not containing chlorides such as KCl and NaCl (that is, metal cations such as K ⁺ and Na ⁺ generated by ionization) is used as a pretreatment liquid, the sensitivity to sweetness It is confirmed that is not obtained.

  FIG. 18 shows the measurement results in the case of using the three types of pretreatment liquid that seems to have high responsiveness and the pretreatment liquid from which the salt (chloride) was omitted.

  From this measurement result, it is understood that practical sensitivity cannot be obtained with the pretreatment liquid containing no chloride in the pretreatment liquid, and that this chloride is greatly involved in the sweetness response.

  In addition, although the measurement results are omitted, for example, iodides (KI, NaI) and bromides (KBr, KI, NaBr) other than the above-mentioned chlorides can be used as pretreatment liquids as long as they have a metal cation. A sufficient response for sweetness detection has been obtained, and it has been confirmed that the sweetness response is not affected by the types of anions such as chlorine, iodine and bromine.

  From these experimental results, it is confirmed that a sweetness response can be obtained by the action of the hydroxyl group of the sugar substance and the metal cation of the pretreatment liquid as described above.

  The membrane sensor of the above embodiment uses trimellitic acid as a weakly acidic substance that is largely involved in the sweetness response. From the specified measurement results shown in FIG. 3, at least the trimellitic acid is presented in FIG. High sensitivity and selectivity with respect to sweetness are also observed for membrane sensors containing weakly acidic substances having carboxyl groups or phosphate groups.

  In this regard, FIG. 3 shows only representative values of the content ratio. However, in actuality, experiments were performed with a larger content ratio, and the sensitivity to sweetness was not limited to the content ratio shown in FIG. And selectivity is recognized.

  Some measurement results are shown in FIGS. These measurement results are responses of the membrane sensor to the 300 mM sucrose solution when the contents of lipid TDAB and weakly acidic substance (sweet responsive substance) are changed. In each case, not only the content ratio shown in FIG. It can be seen that a sufficiently high response is obtained even in the vicinity thereof.

  In this way, high sensitivity and selectivity for low-concentration sweetness (especially high discrimination for umami) can be obtained and response in many of weakly acidic substances having a carboxyl group or a phosphate group and having hydrophobicity. Since the principle is also clear, it is effective for the membrane sensor used for sweetness detection of the present invention as long as it has a carboxyl group or a phosphate group and is hydrophobic even if it is other than the weakly acidic substance shown in FIG. It is.

  DESCRIPTION OF SYMBOLS 100 ... Membrane sensor, 101 ... Tube, 102 ... Membrane body, 103 ... Buffer layer, 104 ... Electrode, 200 ... Reference electrode, 203 ... Buffer layer, 204 ... Electrode, 300 ... Container , 301... Voltage detection circuit, 302... A / D converter, 303.

Claims (4)

A membrane sensor comprising a polymer material, a plasticizer, and a weakly acidic substance having a carboxyl group or a phosphate group and having a hydrophobic portion is immersed in an alkaline pretreatment liquid containing a metal cation. Adsorbing the metal cations on the membrane surface by interaction with the weakly acidic substance of the membrane sensor;
The membrane sensor to which the metal cation is adsorbed is immersed in a liquid to be measured containing a sugar substance, and the metal cation is separated from the membrane surface by interaction with the sugar substance, thereby changing the membrane potential in the negative direction. A sweetness test method comprising the steps of: A polymer material, a plasticizer, and a weakly acidic substance having a carboxyl group or a phosphate group and having a hydrophobic portion are mixed to form a film , and a metal cation interacts with the weakly acidic substance. A film sensor for sweetness testing, wherein the film sensor is adsorbed on the film surface by an action . 3. The membrane sensor for sweetness testing according to claim 2 , wherein the polymer material is contained in a ratio of 800 mg of a polymer material PVC, 1 ml of a plasticizer DOPP, and 30 to 120 mg of a weakly acidic substance trimellitic acid . The membrane sensor for sweetness testing according to claim 3, wherein lipid TDAB is contained at a ratio of 1 mg or less .

Images