JP2811742B2 - Sugar content measurement ink composition and test object - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial applications]

TECHNICAL FIELD The present invention relates to an improvement in a sugar content measurement test body and an ink composition used for the same, which easily quantifies a sugar content in a solution containing a large amount of sugar content such as fruit juice.

[Prior art]

It is convenient if the sweetness can be known in advance in harvesting, selling, and tasting fruits. As a means for quantifying the sugar content in food, a method based on measurement of refractive index or an analytical method using a nuclear magnetic resonance apparatus or high performance liquid chromatography is known. However, these methods require a certain degree of technology and require expensive measuring equipment, and cannot be easily implemented. Therefore, the applicant has developed a test body (Japanese Patent Application Laid-Open No.
No. 62-263468), an ink composition for measuring sugar content and a test piece which are suitable for a high-concentration solution by adding a color reaction inhibitor thereto have been completed, and have already been proposed (Japanese Patent Application No. 63-63).
−18423). This is done by adding a drug, such as sodium bisulfite, that inhibits the action of sugar oxidase and lowers the degree of coloration,
The range in which the sugar content can be determined is shifted to the higher concentration side. Since the sugar content of fruit juice has a wide range from a relatively low value to a considerably high value depending on the type and time of the fruit, it is desirable that a wide concentration range can be quantified by one test object.
If the concentration range that can be measured is narrow, it is necessary to predict the sugar concentration of the sample to some extent and to determine the amount of the color reaction inhibitor added so as to match the sugar concentration. In addition, the sources of sweetness such as fruits are glucose, fructose,
There is also sucrose and the like, and the ratio of their contents varies depending on the degree of maturation. Therefore, even if the sugar content is measured based on the concentration of glucose, it does not always coincide with the sweetness perceived by taste.

[Problems to be solved by the invention]

An object of the present invention is to make it possible to determine not only glucose but also sucrose in addition to glucose among sugars contained in fruits and the like, and that the sugar concentration can be determined over a wide range from medium to high values. An object of the present invention is to provide a sugar content measuring ink composition and a test body as described above.
Furthermore, it is also an object of the present invention to provide an ink composition and a test body that can more quickly measure the sugar content.

[Means for Solving the Problems]

The ink composition for measuring the sugar content of the present invention is obtained by dissolving or dissolving a reagent composition comprising sucrose converting enzyme, glucose oxidase, peroxidase, an oxidizable indicator, and a pH buffer in a solvent, preferably a non-aqueous solvent. Be dispersed. Sucrose invertase is an enzyme that decomposes sucrose into glucose and fructose, and invertase is known to perform this function. Glucose oxidase is an enzyme that oxidizes glucose to gluconic acid and H ₂ O ₂ , and a known glucose oxidase may be used. The amount of these enzymes is 0.004 to 5% by weight, preferably 0.04 to 1% by weight, and the latter is based on the solid content of the ink composition, if the activity is freeze-dried to a titer of 100 unit / mg. Is selected from the range of 0.004 to 5% by weight, preferably 0.04 to 1% by weight. When it is desired to measure the sugar content quickly, a reagent composition containing glucose isomerase in addition to the above components is used. Glucose isomerase is obtained by converting sucrose into α.
-An enzyme that isomerizes anomeric glucose into β-anomeric glucose that can be oxidized by glucose oxidase. Mutarotase is known to perform this function. As with other enzymes, the activity of this enzyme is 100 units /
In terms of a freeze-dried product having a titer of mg, the amount is selected from the range of 0.001 to 0.5% by weight, preferably 0.01 to 0.1% by weight, based on the solid content of the ink composition. Peroxidase is an enzyme that helps oxidize various organic substances with hydrogen peroxide or organic peroxides. When this enzyme is also used as a freeze-dried product having a titer of 100 unit / mg, 0.002 to 3% by weight based on the solid content of the ink composition,
Preferably it is present in an amount of 0.02-0.6% by weight. An oxidizable indicator is an indicator that is oxidized by an enzyme to develop color. The indicator used in the present invention is not selected from high-sensitivity compounds such as benzidines and N-alkylated benzidines, but selected from low-sensitivity compounds such as naphthol derivatives. 1,7-dihydroxynaphthalene is a stable substance and is particularly preferred. This component,
An amount of 0.5 to 10% by weight, preferably 0.6 to 6% by weight, based on the solids content of the ink composition is used. In order to measure the sugar content over a wider range, a mixture of the oxidized naphthol derivative and a naphthol derivative that hardly develops a color even when oxidized may be used as the oxidizable indicator. Examples of naphthol derivatives that hardly develop color even when oxidized include 1,3-dihydroxynaphthalene, 1,4-dihydroxynaphthalene,
1,5-dihydroxynaphthalene, 1,6-dihydroxynaphthalene, 2,4-dichloro-1-naphthol, 1,4-dihydroxyanthraquinone, N-butyl-1-hydroxy-
2-naphthoic amide, and N-dodecyl-1-hydroxy-2-naphthoic amide. Among them, a substance having the following structure, which is a stable substance, ie, N
-Alkyl-1-hydroxy-2-naphthoic amides are preferred. [In the formula, R represents an alkyl group. The above-mentioned substance is used in an appropriate amount depending on the measurement range of the sugar content, and is usually 0.5 to 2 with respect to the solid content of the ink composition.
0% by weight, preferably 0.6-15% by weight. The pH buffer is used to maintain a pH value (for example, pH 5) suitable for the oxidizable indicator to develop a color in the ink composition for sugar detection. Specifically, a combination of citric acid and sodium citrate is preferred. In addition to the above components, it is preferable to use a reagent composition containing a binder, and furthermore, a binder and a water-absorbing powder. The binder is required to have no effect on the components, pH, reagents, especially enzymes and oxidizable indicators in the solution for measuring the sugar content, and not to interfere with the coloring reaction. The binders that have been confirmed to satisfy this condition include (I)
Synthetic resins such as polyester, alkyd resin, polyurethane, polystyrene, acrylic resin, epoxy resin, polyvinyl chloride (including copolymer), polyvinyl butyral, polyvinyl alcohol, polyvinyl pyrrolidone, and maleic anhydride copolymer; ) Methylcellulose, ethylcellulose, hydroxyethylcellulose,
Cellulose derivatives such as carboxymethyl cellulose,
(III) There are natural polymers such as starch, polysaccharides, gelatin, casein, and sodium alginate. These binders may be used in combination of two or more. The binder is used in an amount of 0.1 to 20% by weight, preferably 0.5 to 20% by weight, based on the solid content of the ink composition.
A quantity of ~ 10% by weight may be used. The water-absorbing powder increases the water absorption of the reagent composition provided on the support to provide contact between the liquid to be measured and the reagent composition,
Promotes the color reaction of the indicator. As the water-absorbing powder having such an action, a powder which does not show acidity or alkalinity when contacted with water and has high whiteness is preferable. Specifically, kaolin, synthetic silica, glass, cellulose block, microcrystalline cellulose, ion exchange cellulose, ion exchange resin, calcium carbonate, magnesium carbonate, aluminum silicate and the like are used. The water-absorbing powder preferably accounts for 30 to 90% by weight of the solid content of the ink composition. To prepare the ink composition, each of the above components as a solvent,
Preferably, it is dissolved or dispersed in a non-aqueous solvent containing substantially no water. Examples of such non-aqueous solvents include benzene, aromatic hydrocarbons such as toluene, aliphatic ketones such as methyl ethyl ketone, esters such as ethyl acetate,
Alternatively, alcohols such as n-butanol are used. Among alcohols, C ₁ -C ₂ lower alcohols are not preferred because they cause deactivation of enzymes. In order to make the reagent composition stable, it is preferable to use a stabilizer. The main role of the stabilizer is to prevent the oxidizable indicator from discoloring due to the action of peroxides in the atmosphere. Specifically, a compound having an antioxidant property, a certain surfactant represented by glycerol esters, or a mixture thereof is used. The amount of the stabilizer added is not particularly limited, but is suitably from 0.02 to 5% by weight based on the solid content of the ink composition. In addition to the above components, a wetting agent may be added to the ink composition to facilitate dispersion of each reagent, promote formation of a uniform reagent layer, and improve water wettability. As the wetting material, nonionic surfactants, anionic surfactants, cationic surfactants, amphoteric surfactants, polyethylene glycols, and the like are used. The amount is suitably 0.5 to 5% by weight based on the solid content of the ink composition. Further, a background dye such as oil yellow may be added to make the color tone of the indicator easier to see. The test object for measuring sugar content of the present invention is obtained by applying or impregnating the above ink composition onto a support. Application may be by printing or by coating (eg, roll coating, spray coating, dip coating). In particular, silk screen printing, intaglio printing, and gravure printing are preferred, in which the amount of the ink composition that can be applied is relatively large and the amount of application can be easily made constant. An appropriate coating amount varies depending on the type of the ink composition, but is generally ² to 150 g / m ² (dry amount).
It is. When applying the ink composition, a support that does not react with the reagent composition and does not inhibit the coloration of the reagent is used. Paper, synthetic paper, nonwoven fabric or synthetic resin film, or a laminate of paper and synthetic resin film is used. In addition, paper or nonwoven fabric, particularly filter paper, is preferable as the support when the ink composition is impregnated. The test object of the present invention may be in the form of a stick, a roll, a tape, or the like, in addition to a flat test paper. The support itself may be in a form capable of containing the liquid, for example, a cup, a test tube, a plate, a tray, a dropper, and the test object may be applied thereon.

[Operation]

The ink composition for sugar content measurement and the test body of the present invention decompose sucrose into glucose and fructose with a sucrose converting enzyme such as invertase, and react with a glucose oxidase such as glucose oxidase and an enzyme in the air. , by oxidizing glucose produced by the decomposition of glucose and sucrose contained originally in the sample to produce gluconic acid and H ₂ O _2, reactive the H ₂ O ₂ decomposes peroxidase It utilizes a mechanism that produces high O, which causes the oxidizable indicator to develop color. Therefore, not only glucose but also sucrose is included in the measurement of the sugar content, and a value of the sugar content that is well matched to the sweetness perceived by taste can be obtained. Glucose obtained by decomposing sucrose includes β-anomeric glucose that can be oxidized by glucose oxidase and α-anomeric glucose that cannot be oxidized. Since both are in an equilibrium relationship, when the β-anomeric glucose is no longer oxidized, the α-anomeric glucose gradually isomerizes to β-anomeric glucose. As a result, almost all of the sucrose is ultimately measured as glucose, but it takes some time. That is, since isomerization is rate-determining in the above-described coloration mechanism, the time required for measurement is longer for a sample containing much sucrose than for a sample containing only glucose. Therefore, by using an enzyme such as mutarotase that promotes isomerization of glucose, the step of controlling the coloration mechanism can be rapidly performed, and the sugar content of a sample containing a large amount of sucrose can be measured quickly. . By using a low-sensitivity indicator such as a naphthol derivative as the oxidizable indicator, a relatively low sugar content (higher than when using a high-sensitivity indicator such as guaiac oil, but a color reaction inhibitor) ) Can be quantified over a wide range from those having a high sugar content such as fruit juice of a mature fruit) to those having a high sugar content. Furthermore, when a naphthol derivative that hardly develops a color even when oxidized is used in a mixture at an appropriate ratio, measurement can be performed even in a region having a higher sugar content. Two naphthol derivatives,
In other words, the oxidation of those that are oxidized and those that do not develop a color, although the ratios of the speeds are slightly different depending on the combination, but proceed almost in parallel, so that the effect of the former being diluted by the latter occurs, and the coloration degree is different. Is seen over a wide area.

Comparative Example 1 An ink composition for measuring a sugar content was prepared by pulverizing and dispersing the following composition ("parts" is parts by weight; hereinafter, this is referred to as "reference composition") using a homomixer. Glucose oxidase (“Grade II” manufactured by Toyobo Co., Ltd.) 3.6 parts Peroxidase (“Grade III” manufactured by Toyobo Co., Ltd.) 2.4 parts Guayak butter Sorbitan monolaurate “Span 20” (manufactured by Kao Corporation) 7.2 Part L-ascorbyl stearate 0.48 part citric acid 2.8 parts sodium citrate 11.0 parts polyvinylpyrrolidone "Koridone 90" (manufactured by BASF) 12.6 parts polyvinyl butyral "ESLEC BX-1" (manufactured by Sekisui Chemical Co., Ltd.) 2.25 parts cellulose fine Powder “AVICEL TG-D” (manufactured by Asahi Kasei Corporation) 171 parts n-amyl alcohol 228 parts butyl cellosolve acetate 33.5 parts This ink was screen-printed to a thickness of 300 μm.
5 m on a support consisting of biaxially oriented polystyrene sheet
Printing was performed so as to form an m-square square to obtain a test piece for measuring sugar content. When the test sample was brought into contact with a glucose solution, it exhibited a pale blue color from a solution having a concentration of 0.05 g / dl, and showed a gradation in which the blue color increased according to the sugar concentration up to 2 g / dl. However, no difference in coloration due to the concentration difference was observed for the high concentration solution of 2 g / dl or more.

Comparative Example 2 An ink composition for measuring sugar content was produced in the same manner as in Comparative Example 1, except that an ink composition prepared by adding 7 parts of sodium bisulfite to a reference composition was used. β-D-glucose was diluted with purified water to a concentration of 5 g / dl, 8 g / dl, 13 g / dl, and 20 g / dl, and the test specimen was immersed in them, immediately taken out, and allowed to stand for 5 minutes. Was observed. Those immersed in the 8 g / dl, 13 g / dl and 20 g / dl solutions turned blue and their concentration gradually increased with the increase of the sugar concentration, but they were immersed in the 5 g / dl solution What was done did not change. The coloration was stable for about 30 minutes.

[Reference Example 1] 4.8 parts of guaiac fat was combined with 1,7-dihydroxynaphthalene 4.8 parts.
An ink composition having the same composition as the reference composition was used, except that the composition was changed to parts. This specimen exhibited a pale blue color from a solution having a glucose concentration of 0.5 g / dl, and showed a gradation in which the blue color increased according to the glucose concentration up to 8 g / dl. In particular, in the region from 1 g / dl to 5 g / dl, the difference in gradation for each 1 g / dl could be sufficiently confirmed visually. This coloration was stable for about 30 minutes.

Example 1 The results of tracking changes in the sugar composition in fruit juice after fruiting of watermelon are published as follows. [Cucumber cultivation complete book
(19, III, watermelon, 1973, agricultural book) Malic acid 250mg / dl and citric acid with the same sugar composition as above
An aqueous solution containing 40 mg / dl (pH 5.5) was subjected to a sensory test of sweetness by six panelists using the sequential method (1 for the sweetest and 1 for the weakest). Was. At the same time, a test piece similar to Comparative Example 1 using an ink composition obtained by adding 5 parts of invertase (manufactured by Seikagaku Corporation, 100 unit / mg) to the composition of Reference Example 1 was used. Was measured. The sugar content was determined by comparing with the gradation obtained in Reference Example 1. The test results are as follows. As can be seen from the above results, there is a correlation between the result of the sensory test and the result of the sugar content measurement by the test body.

Reference Example 2 The same test as that of the example was performed using the test piece of Reference Example 1. Although the measurement results were in accordance with the glucose concentration, there was no correlation with the total sugar amount or the results of the sensory test.

[Example 2] Sensory test of sweetness by 15 panelists for four kinds of watermelons from different production areas Measurement of sugar content by Brix refractometer Comparative example 1 using the same test sample as used in Example 1
When the sugar content was measured by comparison with the coloration state, the test results were as follows.The measurement results with the test body of the present invention, the results of the sensory test and the results of the sugar content measurement with the Brix saccharimeter And there was a correlation.

Example 3 The following ink composition for measuring sugar content was prepared, and a test piece for measuring sugar content was obtained in the same manner as in Comparative Example 1. Sucrose invertase ("Grade I" manufactured by Toyobo Co., Ltd.) 11.0 parts Glucose oxidase ("Grade II" manufactured by Toyobo Co., Ltd.) 11.0 parts Peroxidase ("Grade III" manufactured by Toyobo Co., Ltd.) 7.0 parts 1,7-dihydroxynaphthalene 7.0 parts Sorbitan monolaurate “SPAN 20” (manufactured by Kao Corporation) 7.2 parts L-ascorbyl stearate 4.8 parts citric acid 2.8 parts sodium citrate 11.0 parts polyvinylpyrrolidone “Koridone 90” (manufactured by BASF) 12.6 parts Polyvinyl butyral "ESREC BX-1" (manufactured by Sekisui Chemical Co., Ltd.) 2.25 parts Cellulose fine powder "Avicel TG-D" (manufactured by Asahi Kasei Corporation) 171 parts n-amyl alcohol 228 parts Butyl cellosolve acetate 33.5 parts This test specimen shows a pale blue color from a solution with a glucose concentration of 0.5 g / dl when contacted with a glucose solution, and a blue color according to the glucose concentration up to 8 g / dl. It shows a gray-scale increase. In particular, in the region from 1 g / dl to 5 g / dl, the difference in gradation for each 1 g / dl could be sufficiently confirmed visually. The time required for the coloration to become constant was about 2 minutes. Similarly, when the sucrose solution was brought into contact with the sucrose solution, the sucrose solution exhibited a pale blue color from the solution having a sucrose concentration of 1 g / dl, and showed a gradation in which the blue color increased according to the sucrose concentration up to 12 g / dl. It took about 5 minutes for the degree of coloration to become constant.

REFERENCE EXAMPLE 3 A mixed solution containing the same weight of each of 30% aqueous hydrogen peroxide, a 1/15 M phosphate buffer (pH 5) and ethanol was prepared. To 4.5 ml of the above mixture, 5 mg of 1,7-dihydroxynaphthalene, or 5 mg of 1,7-dihydroxynaphthalene and 25 mg
A solution prepared by dissolving a mixture with a naphthol derivative below was prepared. To these solutions, 0.5 ml of a 0.2% peroxidase aqueous solution was added to develop a color, and the absorbance at a wavelength of 600 nm was measured. The results are shown below. Naphthol derivative absorbance (1,7-dihydroxynaphthalene only) 0.41 1,3-dihydroxynaphthalene 0.05 1,4-dihydroxynaphthalene 0.02 1,5-dihydroxynaphthalene 0.20 1,6-dihydroxynaphthalene 0.26 4-chloro-1-naphthol 0.76 1 -Hydroxy-4-methoxynaphthalene 0.98 2,4-dichloro-1-naphthol 0.07 1,4-dihydroxyanthraquinone 0.08 1-hydroxy-2-naphthoic acid methyl ester 0.41 1-hydroxy-2-naphthoic acid phenyl ester 0.42 N-butyl -1-hydroxy-2-naphthoic amide 0.08 N-propyl-1-hydroxy-2-naphthoic amide 0.08 N-dodecyl-1-hydroxy-2-naphthoic amide 0.16

Example 4 Using the ink composition obtained by adding 21 parts of N-propyl-2-naphthoic amide to the composition listed in Example 3, a test piece for measuring the sugar content was produced in the same manner as in Comparative Example 1. did. This specimen exhibited a light blue color from a solution having a glucose concentration of 2 g / dl when contacted with a glucose solution, and showed a gradation of increasing blue according to the glucose concentration up to 14 g / dl. In particular, in the region from 2 g / dl to 10 g / dl, the difference in gradation every 2 g / dl could be sufficiently confirmed visually. The time required for the coloration to become constant was about 2 minutes. Similarly, when the sucrose solution was brought into contact with the sucrose solution, the sucrose solution exhibited a pale blue color from a solution having a sucrose concentration of 3 g / dl, and showed a gradation of increasing the blue color according to the sucrose concentration up to 14 g / dl. It took about 5 minutes for the degree of coloration to become constant.

Example 5 The composition of Example 4 was replaced with mutarotase (Merck, 20 units /
mg) 3.2 parts of the ink composition was used, and a test piece for measuring sugar content was produced in the same manner as in Comparative Example 1. This specimen exhibited a light blue color from a solution having a glucose concentration of 2 g / dl when contacted with a glucose solution, and showed a gradation of increasing blue according to the glucose concentration up to 14 g / dl. In particular, in the region from 2 g / dl to 10 g / dl, the difference in gradation every 2 g / dl could be sufficiently confirmed visually. The time required for the coloration to become constant was about 2 minutes. Similarly, when the sucrose solution was brought into contact with the sucrose solution, the sucrose solution exhibited a pale blue color from a solution having a sucrose concentration of 3 g / dl, and showed a gradation of increasing the blue color according to the sucrose concentration up to 14 g / dl. It took about 2 minutes for the coloration to become constant.

Example 6 Measurement of sugar content using a Brix refractometer for melons having different production areas, varieties and days after harvesting, and a glucose standard solution in Example 5 using the same test body produced in Example 5 The sugar content was measured by comparing with the coloration state when contacted with, and the results of both were compared. The relationship shown in the graph of the figure was obtained, and a high correlation was observed between the measurement result of the test sample of the present invention and the measurement result of the Brix saccharimeter, confirming that the test sample was useful. The time until the coloration was stabilized was about 2 minutes as in the test with the aqueous solution.

【The invention's effect】

INDUSTRIAL APPLICABILITY The ink composition for detecting a sugar content of the present invention and a test body using the same can quickly and easily measure the sugar content of a sugar solution having a high concentration. Therefore, if the sugar content of the fruit is measured with this test object, it can be easily determined whether or not the fruit is ready to eat. This test body has a wide range of measurable sugar content, and it is convenient to preliminarily estimate the sugar concentration of a sample and use an ink composition that matches the sugar concentration.

[Brief description of the drawings]

The drawing is a graph showing the relationship between the sugar content measured by the Brix refractometer and the glucose converted sugar content by the test sample of the present invention, measured in Example 6.

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Claims (10)

(57) [Claims] An ink composition for measuring a sugar content obtained by dissolving or dispersing a reagent composition comprising sucrose converting enzyme, glucose oxidase, peroxidase, an oxidizable indicator, and a pH buffer in a solvent. 2. The ink composition according to claim 1, wherein a reagent composition containing glucose isomerase in addition to the above components is used. 3. The ink composition according to claim 1, wherein a naphthol derivative which develops a color when oxidized is used as the oxidizable indicator. 4. The ink composition according to claim 1, wherein a mixture of a naphthol derivative which develops a color when oxidized and a naphthol derivative which hardly develops a color when oxidized is used as the oxidizable indicator. 5. A naphthol derivative, which hardly develops a color even when oxidized, has the following structure: [In the formula, R represents an alkyl group. The ink composition according to claim 4, wherein 6. The ink composition according to claim 1, wherein a reagent composition containing a binder in addition to the components is used. 7. The ink composition according to claim 6, wherein a reagent composition containing a water-absorbing powder in addition to the components is used. 8. The ink composition according to claim 1, wherein a reagent composition containing a stabilizer in addition to the components is used. 9. The ink composition according to claim 1, wherein a non-aqueous solvent is used as the solvent. 10. A test body for measuring a sugar content obtained by coating or impregnating a support with the ink composition according to any one of claims 1 to 8.