Patent Application
20110262943
The present invention relates to a reagent kit for measuring freshness. The kit can easily measure freshness of, for example, fish meat in a short time and has high storage stability.
Recently, as an index of freshness of, for example, meat, fish meat, or chicken, K value is used. The K value is a ratio (%) of the content of inosine (HxR) and hypoxanthine (Hx) to the total content of adenosine triphosphate (ATP) and ATP decomposition products (ADP, AMP, IMP, HxR, and Hx) in the meat, that is, a numerical value (%) represented by (HxR+Hx)×100/(ATP+ADP+AMP+IMP+HxR+Hx). Herein, ADP denotes adenosine diphosphate, AMP denotes adenosine monophosphate, IMP denotes inosinic acid, HxR denotes inosine, and Hx denotes hypoxanthine.
In order to determine the K value, it is necessary to measure the contents of ATP, ADP, AMP, IMP, HxR, and Hx in meat, fish meat, or chicken. However, the measurement of the contents of these ATP and ATP decomposition products is very troublesome, and in the case of fish meat, ATP, ADP, and AMP are mostly decomposed into IMP (inosinic acid) with time after death of fish, and thereby the amounts of ATP, ADP, and AMP become very low. Accordingly, a numerical value Ki, which is represented by (HxR+Hx)×100/(IMP+HxR+Hx), is assumed as to be Ki≈K and is conveniently used instead of the K value as an index of freshness.
It is known a method for measuring the contents of HxR and Hx as described in, for example, JP-B-62-50120, by mixing a composition solution containing nucleoside phosphorylase (NP), xanthine oxidase (XOD), and a color former with meat juice to decompose HxR in the meat juice to Hx by nucleoside phosphorylase (NP), to decompose Hx to xanthine and uric acid by xanthine oxidase (XOD), and to develop a color by conjugation of the color former with decomposition of Hx, and determining the intensity of the color development.
It is known a method for measuring the contents of IMP, HxR, and Hx as described in, for example, JP-A-Hei-9-262098, by applying a sample solution containing meat juice to a reactor to which alkaline phosphatase (AP), nucleoside phosphorylase (NP), and xanthine oxidase (XOD) are fixed to decompose inosinic acid (IMP) to inosine (HxR) by alkaline phosphatase (AP), to decompose inosine (HxR) to hypoxanthine (Hx) by nucleoside phosphorylase (NP), and to decompose hypoxanthine (Hx) to xanthine and uric acid by xanthine oxidase (XOD), in the sample solution, and measuring the concentration of hypoxanthine (Hx) using a luminous reagent.
However, enzymes (AP, NP, and XOD) used in the above-described methods are very unstable, and their activities easily decrease with time. In addition, their activities further decrease under high temperature. Therefore, it is very difficult to commercialize compositions containing these enzymes.
Against unstableness of these enzymes, for example, JP-A-Hei-8-131196, JP-T-2000-51394, WO 2002/004633, and JP-A-2008-206491 propose some technologies for stabilizing an enzyme by adding sugar (e.g., trehalose or sucrose) or serum albumin (BSA) to an aqueous solution of the enzyme and lyophilizing the solution.
However, there is compatibility between the type of sugar and the type of enzyme for stabilizing the enzyme, and the degree of stabilization of enzyme differs depending on how to combine the enzyme with the sugar. Therefore, the type and concentration of sugar for stabilizing a specific enzyme are not easily determined. In a case of using a mixture of a plurality of enzymes, determination of the type and concentration of sugar for stabilizing these enzymes are further difficult.
When an enzyme is stabilized by freezing an aqueous solution containing the enzyme and then drying it, the conditions, in particular, the conditions in the process for drying the frozen product by increasing the temperature after the freezing also highly affect the stability of the enzyme, but optimum conditions are not known. In a case using a mixture of a plurality of types of enzymes, it is further difficult to determine the optimum conditions.
The problem to be solved is that there is not a reagent kit for measuring freshness, as means for measuring freshness of fish meat, etc., by providing an enzyme activity necessary for appropriately measuring freshness of fish meat, etc. with high storage stability in which enzyme activity is hardly decreased even after storage at high temperature for a long time.
The main characteristics of the reagent kit for measuring freshness according to the present invention are that, in order to enhance the storage stability of enzymes contained in the reagent kit for measuring freshness, an enzyme aqueous solution containing an enzyme protecting agent (sugar and/or gelatin) is quickly frozen, and the resulting frozen product is dried at a temperature not higher than the glass transition temperature (Tg) of the frozen product under reduced pressure.
That is, the reagent kit for measuring freshness according to the present invention is composed of a first reagent and a second reagent. The first reagent is a lyophilizate of a first reagent solution containing xanthine oxidase (XOD), nucleoside phosphorylase (NP), an enzyme protecting agent, and a color former, and the second reagent is a lyophilizate of a second reagent solution containing xanthine oxidase (XOD), nucleoside phosphorylase (NP), alkaline phosphatase (AP), an enzyme protecting agent, and a color former.
Herein, the first reagent solution and the second reagent solution are rapidly frozen with, for example, liquid nitrogen and are then dried under reduced pressure. In this case, since the drying time can be shortened by performing the drying while increasing sample temperature (lyophilizer shelf temperature) with elapse of time, the temperature of the solution is preferably increased with elapse of time during the drying of the solution. However, since the enzyme activity decreases if the solution temperature is increased to a temperature not lower than the glass transition temperature (Tg) of the product obtained by the drying, the sample temperature should be increased in the range not higher than the glass transition temperature (Tg) of the product obtained by the drying.
As the enzyme protecting agent, sucrose and/or gelatin can be used. The concentration of sucrose is preferably in the range of 50 to 400 mM. A sucrose concentration in this range can provide a reagent having high storage stability. The concentration of gelatin is preferably in the range of 0.1 to 2.0 (grams of gelatin/100 mL of reagent solution). A gelatin concentration in this range can provide a reagent having high storage stability.
As the color former, one that develops a color by conjugation with a reaction of decomposing hypoxanthine (Hx) into xanthine and uric acid by xanthine oxidase (XOD) can be used, and examples thereof include known formazan reagents such as tetrazolium blue (TB), nitrotetrazolium blue (nitro-TB), tetrazolium violet (TV), nitroblue tetrazolium (NBT), 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium (MTT), 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulphenyl)-2H-t etrazolium salt (WST-1), 2-(4-iodophenyl)-3-(2,4-dinitrophenyl)-5-(2,4-disulphenyl)-2H-tetrazolium salt (WST-3), 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulphenyl)-2H-tetrazolium salt (WST-8), 2,3,5-triphenyltetrazolium, and 3-(4,5-dimethylthiazol-2-phenyl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium salt (MTS).
The concentration of the color former is preferably in the range of 0.1 to 0.6 mM when the color former is WST-8. A color former concentration in this range can give stable color development. In a color former concentration higher than 0.6 mM, the intensity of color development reaches the peak level, which is economically wasteful. Accordingly, the concentration of 0.6 mM has been determined to be the upper limit, but the concentration may be higher than 0.6 mM.
In each of the first reagent solution and the second reagent solution, the concentration of the xanthine oxidase (XOD) is preferably in the range of 0.1 to 10 U/mL, and the concentration of the nucleoside phosphorylase (NP) is, preferably in the range of 0.05 to 20 U/mL. In the second reagent solution, the concentration of the alkaline phosphatase (AP) is preferably in the range of 5 to 200 U/mL.
The lower limit concentrations of the xanthine oxidase (XOD), the nucleoside phosphorylase (NP), and the alkaline phosphatase (AP) have been determined to be 0.1 U/mL, 0.05 U/mL, and 5 U/ml, respectively, because the stabilities of these enzymes are decreased at concentrations lower than these levels. The upper limit concentrations of the xanthine oxidase (XOD), the nucleoside phosphorylase (NP), and the alkaline phosphatase (AP) have been determined to be 10 U/mL, 20 U/mL, and 200 U/mL, respectively, because the storage stabilities of the reagents are decreased at concentrations higher than these levels.
The first reagent solution and the second reagent solution may or may not contain serum albumin (BSA).
The first reagent solution and the second reagent solution preferably each have a pH of 6.8 to 8.5, in which the above-mentioned enzymes can function. The pH levels of the first reagent solution and the second reagent solution are preferably adjusted in the range of 6.8 to 8.5 with a buffer such as potassium phosphate.
The first reagent solution and the second reagent solution may be each impregnated in paper and then lyophilized to give two types of test papers that can be used for measuring freshness of fish meat, etc.
In the present invention, each reagent is prepared by freezing a reagent solution dissolving the above-described two or three types of enzymes and an enzyme protecting agent, and drying the resulting frozen product under reduced pressure in a temperature range of not higher than the glass transition temperature (Tg). This provides advantages that the storage stability (remaining activity) of each reagent has been increased and that each reagent can be stored at relatively high temperature for a long time.
In addition, since each reagent can be stored at relatively high temperature (for example, 55° C.) over a long period of time, the kit can be used, for example, for raw material quality inspection in seafood processing factories and food factories using seafood products. This provides an advantage that freshness of individual fish can be objectively determined on site.
Furthermore, each reagent can be stored at relatively high temperature (for example, 55° C.) over a long period of time. This provides an advantage that the kit can be simply used for determining freshness of fish in areas not having refrigeration facilities, such as developing countries.
The object of providing a reagent kit for measuring freshness, the storage stability of the kit is high, has been realized by a simple method without reducing basic activities of the enzymes.
Nucleoside phosphorylase (NP) was dissolved in 20 mM potassium phosphate buffer (pH=7.8) to prepare a NP solution. Xanthine oxidase (XOD) was dissolved in 20 mM potassium phosphate buffer (pH=7.8) to prepare a XOD solution. Then, stabilizers contained in the NP solution and the XOD solution were removed by dialysis. Sucrose was dissolved in 20 mM potassium phosphate buffer (pH=7.8) to prepare a sucrose solution. WST-8 (color former) was dissolved in 20 mM potassium phosphate buffer (pH=7.8) to prepare a WST-8 (color former) solution.
Then, all of these NP solution, XOD solution, sucrose solution, and WST-8 (color former) solution were mixed to prepare a first reagent solution. This first reagent solution was put in a 2-mL polypropylene tube and immersed in liquid nitrogen for one minute to rapidly freeze the solution. Then, this polypropylene tube was put in a lyophilizer, in a state that the upper lid was opened, for drying. The drying was performed under a reduced pressure of 3.0×10−2 Torr. On this occasion, the temperature was increased from −40° C. to 5° C. by 5° C. for each 3 hours, subsequently, from 5° C. to 25° C. by 10° C. for each 3 hours. Then, the dried product was taken out from the lyophilizer and was transferred to a desiccator containing P2O5 and stored therein for 7 days for complete dehydration to obtain a first reagent.
Nucleoside phosphorylase (NP) was dissolved in 20 mM potassium phosphate buffer (pH=7.8) to prepare a NP solution. Xanthine oxidase (XOD) was dissolved in 20 mM potassium phosphate buffer (pH=7.8) to prepare a XOD solution. An alkaline phosphatase (AP) solution was prepared. Then, stabilizers contained in the NP solution, the XOD solution, and the AP solution were removed by dialysis. Sucrose was dissolved in 20 mM potassium phosphate buffer (pH=7.8) to prepare a sucrose solution. WST-8 (color former) was dissolved in 20 mM potassium phosphate buffer (pH=7.8) to prepare a WST-8 (color former) solution.
Then, all of these NP solution, XOD solution, AP solution, sucrose solution, and WST-8 (color former) solution were mixed to prepare a second reagent solution. This second reagent solution was put in a 2-mL polypropylene tube and immersed in liquid nitrogen for one minute to rapidly freeze the solution. Then, this polypropylene tube was put in a lyophilizer, in a state that the upper lid was opened, for drying. The drying was performed under a reduced pressure of 3.0×10−2 Torr. On this occasion, the temperature was increased from −40° C. to 5° C. by 5° C. for each 3 hours, subsequently, from 5° C. to 25° C. by 10° C. for each 3 hours. Then, the dried product was taken out from the lyophilizer and was transferred to a desiccator containing P2O5 and stored therein for 7 days for complete dehydration to obtain a second reagent.
Then, 60 mg of the first reagent was dissolved in 1 mL of distilled water, and a fraction of 150 μL of the resulting solution was mixed with 150 μL of squeezed juice of fish meat. The absorbance A of the solution mixture was measured at 454 nm with a spectrophotometer. Then, as shown in
Separately, 60 mg of the second reagent was dissolved in 1 mL of distilled water, and a fraction of 150 μL of the resulting solution was mixed with 150 μL of squeezed juice of fish meat. The absorbance A of the solution mixture was measured at 454 nm with a spectrophotometer. Then, as shown in
Then, Ki value (=A/8) was determined based on (HxR+Hx)×100/(IMP+HxR+Hx)=Ki(%), wherein (HxR+Hx) is the concentration A, and (IMP+HxR+Hx) is the concentration B.
Separately, the Ki value of the same fish meat was determined by high-performance liquid chromatography (HPLC). A correlation between the Ki value determined by the reagent kit of the present invention and the Ki value determined by HPLC was obtained. The results were as shown by the graph in
The reagent kits of the present invention were stored at 5° C., 25° C., or 40° C. for 50 days, and the Ki value of fish meat (Japanese horse mackerel) was measured by each of the kits. The results are shown in
In an experiment as in Example 1, when the concentration of alkaline phosphatase (AP) was less than 5 U/mL, the resulting Ki values were unstable, which may be caused by that decomposition of IMP by alkaline phosphatase (AP) was incomplete, and when the concentration of alkaline phosphatase (AP) was higher than 200 U/mL, the storage stability of the reagent was decreased. However, when the concentration of alkaline phosphatase (AP) was from 5 to 200 U/mL, such disadvantages did not occur to give stable Ki values. Accordingly, the optimum concentration range of alkaline phosphatase (AP) is judged to be from 5 to 200 U/mL.
In an experiment as in Example 1, when the concentration of nucleoside phosphorylase (NP) was less than 0.05 U/mL, the resulting Ki values were unstable, which may be caused by that decomposition of HxR by nucleoside phosphorylase (NP) was incomplete, and when the concentration of nucleoside phosphorylase (NP) was higher than 20 U/mL, the storage stability of the reagent was decreased. However, when the concentration of nucleoside phosphorylase (NP) was from 0.05 to 20 U/mL, such disadvantages did not occur to give stable Ki values. Accordingly, the optimum concentration range of nucleoside phosphorylase (NP) is judged to be from 0.05 to 20 U/mL.
In an experiment as in Example 1, when the concentration of xanthine oxidase (XOD) was less than 0.1 U/mL, the resulting Ki values were unstable, which may be caused by that decomposition of Hx by xanthine oxidase (XOD) was incomplete, and when the concentration of xanthine oxidase (XOD) was higher than 10 U/mL, the storage stability of the reagent was decreased. However, when the concentration of xanthine oxidase (XOD) was from 0.1 to 10 U/mL, such disadvantages did not occur to give stable Ki values. Accordingly, the optimum concentration range of xanthine oxidase (XOD) is judged to be from 0.1 to 10 U/mL.
In an experiment as in Example 1, when the concentration of sucrose was less than 50 mM, the resulting Ki values were unstable, and when the concentration of sucrose was higher than 400 mM, the enzyme solution was crystallized. However, when the concentration of sucrose was from 50 to 400 mM, such disadvantages did not occur to give stable Ki values. Accordingly, the optimum concentration range of sucrose is judged to be from 50 to 400 mM.
In an experiment as in Example 1, when the concentration of WST-8 (color former) was less than 0.1 mM, the color development was unstable, and when the concentration of WST-8 (color former) was higher than 0.6 mM, the degree of color development reached the peak level regardless of an increase in concentration. However, when the concentration of WST-8 (color former) was from 0.1 to 0.6 mM, such disadvantages did not occur to give stable Ki values. Accordingly, the optimum concentration range of WST-8 (color former) is judged to be from 0.1 to 0.6 mM.
In an experiment as in Example 1, when the pH was lower than 6.8 or higher than 8.5, the enzymes did not function, but when the pH was from 6.8 to 8.5, such disadvantage did not occur to give stable Ki values. Accordingly, the optimum pH range is judged to be from 6.8 to 8.5.
In the above-described examples, the color intensity of a reagent was determined using an absorptiometer, but, as shown in
A first reagent solution containing 0.3 U/mL of nucleoside phosphorylase (NP) and 0.6 U/mL of xanthine oxidase (XOD) was prepared by dissolving 0.02 mg of NP and 2 mg of XOD in 1 mL of a solution containing 200 mM sucrose or in 1 mL of a solution containing 200 mM sucrose and 0.5% gelatin.
A second reagent solution containing 45 U/mL of alkaline phosphatase (AP), 0.3 U/mL of nucleoside phosphorylase (NP), and 0.6 U/mL of xanthine oxidase (XOD) was prepared by dissolving 1.5 μL of AP, 0.02 mg of NP, and 2 mg of XOD in 1 mL of a solution containing 200 mM sucrose or in 1 mL of a solution containing 200 mM sucrose and 0.5% gelatin.
One milliliter of the first reagent solution was put in a 2-mL polypropylene tube, and 1 mL of the second reagent solution was put in another 2-mL polypropylene tube. These polypropylene tubes were immersed in liquid nitrogen for at least one minute to rapidly freeze the solutions. The resulting frozen products were stored at −90° C.
Then, each frozen product was transferred to a lyophilizer cooled in advance to −40° C., and the temperature was increased from −40° C. to 5° C. by 5° C. for each step, subsequently, from 5° C. to 25° C. by 10° C. for each step, while maintaining the temperature in each step for 3 hours, to dry the frozen product. In this procedure, the pressure in the lyophilizer was maintained at 3.0×10−2 Torr over the drying process. The frozen product lost its water by the lyophilization to form a dried solid.
The remaining water in the dried solid was further removed in a vacuum desiccator over P2O5 at room temperature for 7 days. The dried solid obtained from the frozen product of the first reagent solution and the dried solid obtained from the frozen product of the second reagent solution were placed in a glove box replaced with dried nitrogen as a first reagent and a second reagent, respectively, and were stored at 25° C., 40° C., or 55° C. for 45 days.
Then, 60 mg of the first reagent was dissolved in 1 mL of distilled water. To a fraction of 75 μL of the resulting solution, 75 μL of 4 mM HxR and then 150 μL of 20 mM potassium phosphate buffer were added to prepare a first reagent solution. The absorbance at 292 nm of the first reagent solution was measured at 20° C. using a UV-VIS spectrophotometer. The enzyme activity was evaluated by comparing it with the initial activity.
Similarly, 60 mg of the second reagent was dissolved in 1 mL of distilled water. To a fraction of 75 μL of the resulting solution, 75 μL of 4 mM HxR and 1 mM MgCl2 and then 150 μL of 20 mM potassium phosphate buffer were added to prepare a second reagent solution. The absorbance at 292 nm of the second reagent solution was measured at 20° C. using a UV-VIS spectrophotometer. The enzyme activity was evaluated by comparing it with the initial activity. The remaining activity was expressed by percentage with respect to the activity before the lyophilization.
The remaining activities of the enzymes in the first reagent stored at a storage temperature of 25° C. for from 0 to 45 days were as shown in
It can be confirmed from the results shown in
A first reagent containing sucrose and gelatin and a second regent containing sucrose and gelatin were prepared and were stored at 55° C. for from 0 to 45 days in a relative humidity environment of 0%, 33%, or 53%. The remaining activity of each reagent in each humidity environment was investigated on each day of the storage period. Herein, each reagent was produced by the same method as in Example 10, and the remaining activity of each reagent was measured by the same method as in Example 10.
The remaining activity of the first reagent in each humidity environment was as shown in
A first reagent not containing sucrose and gelatin, a first reagent (Tg: 65° C., MT: 0.81%) containing sucrose but not containing gelatin, and a first reagent (Tg: 77° C., MT: 0.5%) containing both sucrose and gelatin were prepared and stored at 5° C., 25° C., or 40° C. for from 0 to 112 days.
A second reagent not containing sucrose and gelatin, a second reagent (Tg: 65° C., MT: 0.78%) containing sucrose but not containing gelatin, and a second reagent (Tg: 76° C., MT: 0.99%) containing both sucrose and gelatin were prepared and stored at 5° C., 25° C., or 40° C. for from 0 to 112 days. Herein, the fundamental compositions and conditions for preparing the first reagents and the second reagents were the same as those in Example 10, and a color former, WST-8, was added to each reagent.
Inosine (HxR) standard solutions were prepared by adding 0, 5, 10, 20, 30, or 50 μM HxR to 20 mM potassium phosphate buffer. Inosinic acid (IMP) standard solutions were prepared by adding 0, 5, 10, 20, 30, or 50 μM IMP and 1 mM MgCl2 to 20 mM potassium phosphate buffer. The actual HxR concentrations in the HxR standard solutions and the actual IMP concentrations in the IMP standard solutions were determined by high-performance liquid chromatography (HPLC).
The first reagent solution (150 μL) was added to 150 μL of each of the HxR (0, 5, 10, 20, 30, and 50 μM) standard solutions, and the absorbance at 454 nm of each solution was measured with a UV-VIS spectrophotometer at 25° C. to determine a relationship between the HxR concentration and the absorbance, and a standard curve was prepared based on the relationship. The second reagent solution (150 μL) was added to 150 μL of each of the IMP (0, 5, 10, 20, 30, and 50 μM) standard solutions, and the absorbance at 454 nm of each solution was measured with a UV-VIS spectrophotometer at 25° C. to determine a relationship between the IMP concentration and the absorbance, and a standard curve was prepared based on the relationship.
Fish meat (1 g) was mixed with 10% perchloric acid (4 mL) while mincing, and 5% perchloric acid (4 mL) was further added thereto and mixed. The resulting mixture was centrifuged at a speed of 2000 rpm at 5° C. for 10 minutes. The supernatant was collected and was adjusted its pH to 6.8 to 7.0 with 8 M or 1 M KOH. The supernatant was centrifuged again at a speed of 2000 rpm at 5° C. for 10 minutes. The sample solution was filtered through Watman No. 1 filter paper and diluted with distilled water graded by high-performance liquid chromatography (HPLC) to obtain 20 mL of a fish extract. The diluted sample solution was filtered again through millipore paper with a pore size of 0.65 μm and stored at −90° C. until measurement of freshness.
Then, the Ki values of the same fish meat sample on each day of the storage period and at each storage temperature were investigated using the first reagent and the second reagent. Herein, the Ki value of the fish meat sample was determined as follows: First, the first reagent and the second reagent (60 mg each) were each dissolved in 1 mL of distilled water, and to 150 μL of each of the resulting solutions, the fish extract (20 μL) and 20 mM potassium phosphate buffer (130 μL) were added to prepare a first reagent solution and a second reagent solution.
The absorbance at 454 nm of each of the first reagent solution and the second reagent solution was measured with a UV-VIS spectrophotometer at 25° C., and the concentration of HxR+Hx and the concentration of IMP+HxR+Hx were respectively determined using the results obtained by the first reagent solution and the second reagent solution based on the standard curves. Then, the Ki value was determined by fitting the concentration of HxR+Hx and the concentration of IMP+HxR+Hx to the equation shown by the following Expression 1:
Ki(%)=(HxR+Hx)×100/(IMP+HxR+Hx) [Expression 1]
The Ki values determined using the enzyme composite (first reagent and second reagent) not containing sucrose and gelatin were as shown in
It can be confirmed from the results shown in
| Number | Date | Country | Kind |
|---|---|---|---|
| 2008-268871 | Oct 2008 | JP | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/JP2009/067502 | 10/7/2009 | WO | 00 | 7/5/2011 |
