JP4868609B2 - Substrate for measuring proteolytic enzyme activity in salmon, proteolytic enzyme activity measuring method and kit - Google Patents

Description

  The present invention relates to a substrate for measuring proteolytic enzyme activity, a method for measuring proteolytic enzyme activity, and a kit for use in the production of sake, shochu, miso, vinegar and other brews made from rice.

  In the production of brewed products made from rice such as sake, shochu (rice shochu, rice shochu, barley shochu, soba shochu), miso, vinegar (rice vinegar), koji (Aspergillus) is used, Amino acids and peptides are produced from the raw material protein by the proteolytic enzyme, and these amino acids and peptides become important components as taste ingredients. For this reason, since the proteolytic enzyme activity greatly affects the quality of the brewed product, the proteolytic enzyme activity of the koji used has long been measured.

There are two proteolytic enzymes: an endoprotease that breaks down peptide bonds in the middle of high molecular protein chains into low molecular weight soluble proteins and peptides, and an exoprotease that breaks down amino acids one by one from the end of the protein. It is divided roughly into. There are two types of exoproteases: carboxypeptidases that degrade from the C-terminus of proteins and aminopeptidases that degrade from the N-terminus.
Therefore, the proteolytic enzyme activity of koji is acidic in sake and shochu brewing. For example, milk casein is used as a substrate for the activity of acid protease (APase), which is an endoprotease having an optimum pH for acidity. A method of measuring is described in Non-Patent Document 1 below. Patent Document 1 below describes a method of measuring using Cbz-Glu-Tyr as a substrate for the activity of acid carboxypeptidase (ACPase), which is an exoprotease.

It is also possible to use extracted rice protein as a substrate for measuring rice proteolytic enzyme activity. Rice proteins are classified into easily digestible proteins such as glutelin and globulin, and indigestible protein prolamin. Proteins that dissolve in dilute acid or alkaline solutions are dissolved in glutelin, proteins that dissolve in salt solutions are dissolved in globulin and alcohol solutions. Is defined as prolamin.
For example, Patent Document 2 below describes a method in which raw rice is steamed and diluted with acid to extract rice glutelin. However, impurities such as amino acids exist and are not suitable as a substrate for measuring proteolytic enzyme activity. It was appropriate.
Japanese Patent No. 3667470 Japanese Patent No. 3146344 Annotation Editorial Committee, “Fourth Amendment to the National Tax Agency Preliminary Analytical Method,” Japan Brewing Association, 1993

However, in the methods of Non-Patent Document 1 and Patent Document 1, since the substrate is different from the rice protein, the activity of the proteolytic enzyme of koji does not reflect the amount of amino acid produced during brewing. In particular, the substrate for ACPase activity is a single synthetic peptide, showing only the degradation activity from the C-terminal of the protein, and the degradation activity from the N-terminus is ignored.
In the method of Patent Document 2, impurities such as peptides, amino acids, sugars, inorganic salts, and phytic acid are present in the rice extract, and there is a problem as a substrate for accurately measuring the proteolytic enzyme activity of koji. It was.
As described above, there has been no substrate, proteolytic enzyme activity measuring method, and kit that accurately reflects the correlation between the amount of amino acid produced in the brew, which is important for quality, and the total proteolytic enzyme activity of koji.

  The present invention has been made in view of such problems, and an object thereof is to provide a substrate for measuring proteolytic enzyme activity, a method for measuring proteolytic enzyme activity, and a kit that can solve the above-described problems.

In order to solve the above problems, the present invention has the following configuration.
The substrate for measuring proteolytic enzyme activity according to the present invention is a substrate for measuring proteolytic enzyme activity mainly composed of rice glutelin, and the rice glutelin is not defatted and does not steam, to rice powder, rice, rice bran or white rice bran 0.05 to 1 M lactic acid (pH 2.1 to 2.8) was added, stirred, left to stand for 1 to 3 hours, filtered, and rice obtained by isoelectric precipitation at pH 6.4 to 10.0. It is a protein.
The method for producing a substrate for measuring proteolytic enzyme activity according to the present invention is a method for producing a substrate for measuring proteolytic enzyme activity comprising rice glutelin as a main component, wherein the rice glutelin is not defatted and does not steam. , Add 0.05 to 1M lactic acid (pH 2.1 to 2.8) to rice bran or white rice bran, stir for 1 to 3 hours and filter, isoelectric at pH 6.4 to 10.0 It is the rice protein acquired by the point precipitation.
The substrate for measuring proteolytic enzyme activity of the present invention is characterized by being manufactured from the above-described method for producing a substrate for measuring proteolytic enzyme activity.
In the method for measuring proteolytic enzyme activity of sputum according to the present invention, a substrate adjusting step for adjusting the substrate for measuring the proteolytic enzyme activity, an enzyme solution adjusting step for adjusting the sputum enzyme solution, and a substrate adjusted in the substrate adjusting step And an enzyme reaction step for reacting the enzyme solution adjusted in the enzyme solution adjustment step.
In the method for measuring proteolytic enzyme activity of sputum according to the present invention, the enzyme reaction step is performed at pH 3-5.
In the kite proteolytic enzyme activity measurement kit of the present invention, the substrate adjusting means for adjusting the substrate for measuring the protease activity, the enzyme solution adjusting means for adjusting the salmon enzyme solution, and the substrate adjusted by the substrate adjusting means And an enzyme reaction means for reacting the enzyme solution adjusted by the enzyme solution adjustment means.

  According to the present invention, by using a substrate mainly composed of glutelin, a protein extracted from rice as a raw material for brewing, for proteolytic enzyme activity measurement, the proteolytic enzyme activity of koji and the amount of amino acids produced in the brewed product It is possible to perform a measurement that accurately reflects the correlation with.

<Adjustment of rice glutelin substrate>
Embodiment 1 FIG.
Rice glutelin is extracted at pH 3.0 or lower by adding acid to rice, rice bran or white rice. Preferably, rice glutelin is added to 0.05 to 1M lactic acid (pH 2.1 to 2.8) and stirred with respect to rice, rice bran or white rice bran and left to stand for at least 1 hour, preferably 2 to 3 hours. Extract. And after obtaining an extract by filtration or centrifugation (1,000-15,000 rpm, 1-30 minutes), it neutralizes with pH 6.4-10.0 which is the isoelectric point of rice glutelin, Isoelectric point precipitation of rice protein based on glutelin. The main component means that rice glutelin is a major constituent of the whole rice protein when the protein is separated using SDS-PAGE or the like. Then, the precipitate is obtained by centrifugation (1,000 to 15,000 rpm, 1 to 30 minutes), distilled water is added to this to wash out the contaminants adhering to the precipitate, and the precipitate is recovered by centrifugation again. To do. After performing this washing operation 1 to 3 times, the precipitate is lyophilized in vacuum to obtain rice glutelin powder.

  Hereinafter, the preparation of the rice glutelin substrate of the present invention will be specifically described with reference to examples. The present invention is not limited to the following examples, and usual modifications can be made in the technical field of the present invention.

Example 1. Examination of isoelectric point of rice glutelin The sample for electrophoresis was prepared by taking one grain of raw rice in a 1.5 ml microtube, adding 700 μl of swelling / sample buffer (Bio-rad), and slowly stirring at room temperature for 3 hours. Thereafter, centrifugation was performed at 13,000 rpm for 10 minutes, and the supernatant was collected and adjusted.
One-dimensional electrophoresis was performed using a PROTEAN IEF cell (Bio-Rad). IPG Ready Strip, pH 3.0 to 10.0, 11 cm (Bio-Rad) (IPG strip) was set on a focusing tray, 185 μl of a sample for electrophoresis was added, and the mixture was swollen at 50 V for 20 hours. Next, electrophoresis was performed at 8,000 V so as to be 25,000 V / hour.
For the two-dimensional electrophoresis, a Criterion cell (Bio-Rad) was used. The IPG strip was treated with equilibration buffer I and equilibration buffer II (Bio-Rad), and then electrophoresed at 200 V for 60 minutes.
Gel staining was performed with Bio-Safe CBB G-250 (Bio-Rad). The gel was analyzed using GS-800 Calibrated Densitometer (Bio-Rad), and then analyzed using two-dimensional electrophoresis gel image analysis software PDQuest (Bio-Rad).

  The results of examining the isoelectric point of the raw rice protein by two-dimensional electrophoresis are shown in FIG. From these results, the acidic subunit of glutelin is around pH 6.4 to 9.7, the basic subunit of glutelin is around pH 9.7 to 10.0, the globulin is around pH 6.0 to 7.3, and the prolamin is pH 7. It became clear that it has an isoelectric point near 1 to 10.0.

Example 2 Examination of conditions for adjustment of rice glutelin substrate Since rice protein is not commercially available, adjustment of the substrate was examined. First, protein extraction conditions from white rice powder (Yamada Nishiki 55% polished rice) were examined. White rice powder was prepared by grinding white rice in a mill for several minutes. Add 10 ml of various concentrations of lactic acid solution to 1 g of white rice powder and let stand for 1 hour. Filter the filtrate through a 0.45 μm filter, determine the absorbance at 280 nm or Bradford method, and measure the pH of the extract. Went.
The results of examining the extraction conditions of rice protein from rice are shown in Table 1. When the lactic acid concentration was 0.05 M, the pH was 2.8, and the amount of extracted BSA equivalent protein exceeded 2 mg / ml. This concentration almost coincided with the amount of lactic acid used when brewing the sake mother in sake brewing (500 to 700 ml of lactic acid was used in 100 liters of the charged water) and reflected the brewing conditions. Moreover, when the lactic acid concentration was further increased, the amount of extracted protein further increased.

  Next, Table 2 shows the results of examining the influence of the extraction time. The extraction time reached a maximum at 2 hours.

Next, Table 3 shows the results of comparison between stationary and shaking as extraction methods. It was found that there was no significant difference between standing and shaking, and that standing was sufficient.

Next, Table 4 shows the results of examining differences in the amount of extracted protein due to differences in raw rice varieties. There were considerable differences in the amount of extracted protein depending on the variety.

  Next, isoelectric focusing of a rice protein solution extracted from white rice powder with 0.1 M lactic acid was performed. When the extracted protein solution was neutralized with 1N NaOH, a precipitate started to be observed around pH 6.0, and a large amount of precipitate was observed as soon as the pH was further increased to pH 9.0. From this, it was considered that almost all rice glutelin co-precipitated and precipitated around pH 6.5.

  Next, the result of having investigated the rice protein extracted from the white rice powder by SDS-PAGE is shown in FIG. The acrylamide concentration of SDS-PAGE was 15%, and staining after electrophoresis was performed using CBB-R250. Lane M is a marker protein (GE Life Science), Lane 1 is a rice protein extracted using 8M urea, and Lane 2 is extracted using 0.1M lactic acid as described above, and is isoelectric at around pH 6.5. Rice protein that had been point precipitated and lyophilized to powder was used as a sample. From the result of lane 2, when lactic acid extraction was performed, acidic subunits and basic subunits of rice glutelin were extracted almost as main components.

  Since glutelin is dissolved as a weak acid or weak base, globulin is dissolved in a salt solution, and prolamin is defined as a protein that is dissolved in an alcohol solution, first, in the present invention, only rice glutelin was dissolved by acid extraction. Next, obtain only rice glutelin as the main component by isoelectric point precipitation around pH 6.5 except for substances other than rice glutelin, except mainly amino acids, sugars, inorganic salts, phytic acid, etc. (Fig. 2, lane 2). From this, freeze-dried and powdered rice glutelin obtained in this example was used as a substrate for proteolytic enzyme activity of koji.

Example 3 Example of preparation of rice glutelin substrate using Nishida Yamada 55% polished rice Add 5 liters of 0.1M lactic acid to 1kg of white rice powder of Yamada Nishiki 55% polished rice, let stand at room temperature for 2 hours, and centrifuge (3,000rpm, 15 minutes), an extract was obtained. And it neutralized with 1N NaOH, and isoelectric point precipitation was performed at about pH6. Then, centrifugation (5,000 rpm, 15 minutes) was performed, the precipitated solid was suspended in a small amount of distilled water, and the deposits were washed. This operation was repeated twice. Further, centrifugation (5,000 rpm, 15 minutes) was performed, and the precipitated solid was suspended in a small amount of distilled water and freeze-dried. Finally, 7.6 g of rice glutelin powder was obtained from 1 kg of white rice powder of 55% polished rice from Yamada Nishiki. The rice glutelin substrate was prepared by accurately weighing 200 mg of rice glutelin powder, adding 50 ml of a 10 mM lactic acid solution, and 20 mg of benzoic acid as a preservative, and dissolving by heating. The substrate concentration was 4 mg / ml, and the adjusted substrate solution was stored refrigerated.

<Measurement of total proteolytic enzyme activity using rice glutelin as a substrate>
Embodiment 2. FIG.
The total proteolytic enzyme activity measurement method using rice glutelin as a substrate is performed. FIG. 3 is a process diagram of proteolytic enzyme activity measurement of sputum according to the present embodiment.

(1) Substrate adjustment step S1: Preparation of a substrate mainly composed of rice glutelin Embodiment 1 The same adjustment as the rice glutelin substrate described in 1) is performed.

(2) Enzyme solution adjustment step S2: adjustment of proteolytic enzyme derived from cocoon National Tax Agency predetermined analysis method (Reference URL: http: // www. The enzyme solution is prepared according to the stationary extraction method of /pdf/211.pdf). Specifically, 50 ml of sodium chloride solution is added to 10 g of solid soot, and leached while being shaken occasionally in a low temperature room (5 ° C. or less) overnight or at room temperature (15 to 20 ° C.) for 3 hours. 10 ml of the filtrate is put into a dialysis membrane, dialyzed overnight against 0.01 M acetate buffer at a low temperature, and then made up to 20 ml with water to make a sputum enzyme solution.

(3) Enzyme reaction step S3: Reaction between the substrate adjusted in the substrate adjustment step S1 and the enzyme solution adjusted in the enzyme solution adjustment step S2 A buffer solution, preferably a lactic acid buffer, on the rice glutelin substrate adjusted in the substrate adjustment step S1 Liquid pH 3.0-5.0 is added, and it preheats at 30-50 degreeC, Preferably it is 40 degreeC. Then, the dialyzed sputum enzyme solution is added to start the enzyme reaction. The enzyme reaction is performed for 5 to 40 minutes, preferably 10 minutes, 30 to 50 ° C., preferably 40 ° C., and the reaction is stopped by adding trichloroacetic acid (TCA) as a reaction stop solution. And it filters and obtains a reaction liquid.
The substrate blank solution is prepared by replacing the above-described sputum enzyme solution with distilled water. The enzyme blank solution is prepared by replacing the above-mentioned rice glutelin substrate solution with distilled water.

(4) Measuring step S4: Measuring the amount of amino acid obtained in the enzyme reaction step The ninhydrin reagent is added to and mixed with the above reaction solution, substrate blank solution, and enzyme blank solution, and a color reaction is performed at 80 to 100 ° C. Thereafter, the absorbance is measured at 570 nm.
A calibration curve is prepared by adding a ninhydrin reagent to an arginine solution as a standard solution and performing a color reaction, and adding a ninhydrin reagent to distilled water as a control solution and performing a color reaction.
The total proteolytic enzyme activity is defined as 1 unit of activity that produces 1 mg of arginine in 60 minutes. The calculation formula is shown below.

  In the substrate adjustment step S1, rice protein is extracted under acidic conditions of lactic acid, but any acid can be used as long as acidic conditions can be created. For example, the solution may be acidified using an acid such as lactic acid, malic acid, tartaric acid, citric acid, succinic acid, adipic acid, fumaric acid, gluconic acid, phosphoric acid, acetic acid, hydrochloric acid, sulfuric acid or nitric acid. Preferably, the extraction of rice protein is performed at a pH of 3 or less using lactic acid at a concentration of 0.05 M or more. In this case, the protein extracted from rice mainly contains rice glutelin.

  In the substrate adjustment step S1, rice protein precipitation is performed by isoelectric precipitation at the isoelectric point (pH 6.4 to 10.0) of rice glutelin, and the rice protein containing rice glutelin as a main component is collected. Any protein precipitation and concentration methods that can be used by one skilled in the art can also be used. Examples include ammonium sulfate precipitation, ultrafiltration, electrophoresis, density gradient centrifugation, molecular sieve, ion exchange chromatography, affinity chromatography, and chemical precipitation.

  In addition, the rice glisterin may be a recombinant protein expressed in full length or in part. Production of recombinant rice glutelin protein is performed using a general method that can be used by those skilled in the art. The gene sequence of rice glutelin can be obtained from, for example, the Genbank / DDBJ / EMBL database. Rice glutelin is a rice storage protein that forms a multigene family group. For example, rice glutelin-1 is available at http: // www. ncbi. nlm. nih. gov / entrez / viewer. fcgi? It is possible to obtain a gene sequence from db = protein & id = 556399. A full-length or partial-length cDNA encoding rice glutelin protein may be incorporated into a vector and expressed in a host cell such as Escherichia coli, yeast, or cultured cell and recovered. For recovery, any of the protein precipitation methods and concentration methods described above can be used.

  In addition, in the enzyme solution adjustment step S2, adjustment of the proteolytic enzyme derived from sputum is performed according to a method prescribed by the National Tax Agency. For example, homogenization extraction, chemical treatment using a surfactant, or the like may be used.

In the enzyme reaction step S3, it is preferable to adjust the pH using lactic acid (pH 3.0 to 5.0). For example, in the analysis method prescribed by the National Tax Agency, amylase activity is used for acetate buffer, and proteolytic enzyme is used. A citrate buffer solution (Makilben buffer solution) is employed, and these may be used. The enzyme reaction is affected by the enzyme concentration, substrate concentration, inhibitor and activator concentration, pH, temperature, various salt concentrations, etc. You may carry out in consideration of conditions.
In the enzyme reaction step S3, TCA is used as the reaction stop solution, but it is also possible to use, for example, perchloric acid (PCA).

In the measurement step S4, the ninhydrin method, which is a quantification method having high sensitivity to all amino acids and low sensitivity to proteins, is used, but a method for quantifying amino acids that can be used by those skilled in the art can be used. For example, there is a method using a fluorescent reagent.
Further, the total proteolytic enzyme activity is defined as 1 unit of activity for producing 1 mg of arginine in 60 minutes, but can be changed to any activity unit that can be used by those skilled in the art. For example, the activity for producing a single amino acid such as tyrosine or proline may be 1 unit, and the activity for producing a complex amino acid may be 1 unit.

  The total proteolytic enzyme activity measurement using the rice glutelin of the present invention as a substrate will be specifically described below with reference to examples. The present invention is not limited to the following examples, and usual modifications can be made in the technical field of the present invention.

Example 4 Examination of conditions for measuring total proteolytic enzyme activity using rice glutelin as a substrate FIG. 4 shows the results of examining the effect of pH on enzyme reaction using a rice glutelin substrate. The pH was adjusted using 0.1 M Mykilubein buffer. The vertical axis represents the color value of the ninhydrin reaction in terms of arginine.
In the case of the conventional method using milk casein and Cbz-Glu-Tyr as substrates, the optimum reaction pH was 3.0. However, when rice glutelin was used as the substrate, the optimum reaction pH was 4.0 and the sake brewing Consistent with pH range 3.5-4.5. From this result, when measuring the proteolytic enzyme activity of rice bran using rice glutelin as a substrate, it was judged that it is appropriate to perform the pH range around 3.0 to 5.0.
In the conventional method using milk casein as a substrate, the reaction is stopped by adding 0.4 M trichloroacetic acid (TCA) after reacting for a predetermined time, and the unreacted substrate is precipitated and filtered. The amount of TCA soluble protein produced therein is measured and used as the enzyme activity. In this example, as in the conventional method, the substrate was changed to milk casein and rice glutelin was used. After reacting for a predetermined time, the unreacted substrate was removed by precipitation by adding TCA, and the amount of amino acids in the filtrate was measured. Total proteolytic enzyme activity was measured. The ninhydrin method was used for quantification of amino acids.
Table 5 shows the results of examining the effect of temperature on the enzyme reaction. The amino acid production rate (μg arginine / min) reached a maximum at 45 ° C. In view of this result and the effect of enzyme deactivation, it was determined that 40 ° C. was appropriate as the measurement temperature.

  The enzyme degradation pattern of rice protein is shown in FIG. The horizontal axis represents the enzyme reaction time, and the vertical axis represents the amino acid amount (μg arginine) by the ninhydrin color reaction. The amount of amino acid increased linearly in proportion to time up to 40 minutes. From this result, it was judged that a reaction time of 10 minutes was appropriate.

Example 5 FIG. Measurement of total proteolytic enzyme activity using rice glutelin as a substrate (1) Substrate preparation step S1: Preparation of substrate containing rice glutelin as a main component 100 g of white rice powder was extracted with 250 ml of 0.1 M lactic acid for 2 hours. Filtered with 5A. The mixture was neutralized (pH 6.5) with 1N NaOH solution and subjected to isoelectric precipitation. The precipitate was collected by centrifugation (3,000 rpm, 15 minutes), the adhering matter was washed with distilled water, and centrifuged again to obtain a precipitate. This precipitate was freeze-dried to obtain rice glutelin. Then, 0.2 g of rice glutelin powder was weighed, 50 ml of 10 mM lactic acid was added and dissolved by heating to obtain a substrate solution.
(2) Enzyme Solution Preparation Step S2: Preparation of koji-derived proteolytic enzyme Extraction and dialysis were performed according to a method prescribed by the National Tax Agency. 10 g of koji was weighed, 50 ml of koji extract was added, and extracted at room temperature for 3 hours. Toyo Filter Paper No. After filtration with 5A, 20 ml of the filtrate was put in a dialysis tube and dialyzed overnight against 20 mM acetate buffer (pH 5.0). The dialysate was made up to a volume of 40 ml to prepare an enzyme solution.
(3) Enzyme reaction step S3: Reaction of substrate adjusted in substrate adjustment step S1 and enzyme solution adjusted in enzyme solution adjustment step S2 0.1 M lactic acid buffer in 1.0 ml of rice glutelin solution (4 mg of rice glutelin) 1.0 ml of the solution (pH 4.0) was added and preheated at 40 ° C. for 2 minutes. Then, 0.2 ml of dialyzed sputum enzyme solution was added to initiate the reaction. After the reaction for 10 minutes, 3.0 ml of 0.4M TCA solution was added to stop the reaction. A reaction solution was obtained by filtration.
The substrate blank solution was filtered by adding 1.0 ml of 0.1 M lactate buffer (pH 4.0), 0.2 ml of distilled water and 3.0 ml of 0.4 M TCA solution to 1.0 ml of rice glutelin solution. And created. The enzyme blank solution was prepared by adding 0.2 ml of enzyme solution to 1.0 ml of distilled water and 1.0 ml of 0.1 M lactate buffer (pH 4.0), and immediately adding 3.0 ml of 0.4 M TCA solution. Was made without filtration.
(4) Measurement step S4: Measurement of the amount of amino acid obtained in the enzyme reaction step 1.0 ml of ninhydrin reagent (Hitachi amino acid analysis reagent) to 1.0 ml of reaction solution (substrate blank solution, enzyme blank solution, control solution, standard solution) Were added and mixed, and a color reaction was carried out in boiling water for 15 minutes. Thereafter, 5 ml of 60% ethanol was added and stirred, and the absorbance was measured at 570 nm.
The calibration curve was obtained by adding 1.0 ml of ninhydrin to 1.0 ml of arginine (50 μg / ml) solution as a standard solution, and coloring by adding 1.0 ml of ninhydrin to 1.0 ml of distilled water as a control solution. Created by reaction.

Example 6 Correlation between enzyme activity using rice glutelin as substrate and amino acid content of steamed rice digestive juice As for enzyme activity of 33 sake mashes, measurement of proteolytic enzyme activity using rice glutelin of this embodiment as substrate (this method) and as specified by the National Tax Agency FIG. 6 shows the result of comparison with the measurement of proteolytic enzyme activity (conventional method) using the synthetic peptide Cbz-Glu-Tyr as a substrate of the analytical method. The horizontal axis represents the acid carboxypeptidase activity (ACPase) according to the conventional method, and the vertical axis represents the total peptidase activity (total proteolytic enzyme activity) using rice glutelin of this embodiment as a substrate. Thus, even with the same activity in the conventional method, a large difference was observed in the total proteolytic enzyme activity of this method using rice glutelin as a substrate.
Next, an enzyme extract was prepared using 15 koji made by kneading 3 times using 5 koji rice varieties, and a digestion test of five types of steamed rice was carried out. The digestion test was conducted at a temperature of 15 ° C. for 14 days in consideration of the conditions of drought at the actual manufacturing site. Table 6 shows the correlation between the amount of amino acids in the digested rice digestion fluid and the protease activity. Table 6 shows that there were many amino acids that were hardly correlated with the conventional method. The activity was correlated with all amino acids at a risk rate of 5%, and for each amino acid, alanine (r = 0.653 ^** ), glutamic acid (r = 0.766 ^*** ), asparagine related to the taste of sake. acid (r = ^{0.795 ***),} arginine (r = 0.711 ^**) is a high correlation was observed. The branched amino acid (BCAA), which is regarded as an important amino acid in sports drinks, was not correlated with the conventional method, but with this method, a high correlation with the enzyme activity (r = 0.800 ^*** ) was observed. It was. The abundance of essential amino acids was not correlated with the conventional method, but a high correlation coefficient (r = 0.778 ^*** ) was recognized with the enzyme activity in this method.

  From the above results, it is clear that proteolytic enzyme activity using rice glutelin as a substrate shows the activity of many enzymes involved in the degradation of steamed rice protein and has a high correlation with the amount of amino acids produced during brewing. became.

Embodiment 3 FIG. Proteolytic enzyme activity measurement kit FIG. 7 is a conceptual diagram of a kite proteolytic enzyme activity measurement kit according to the present embodiment.
A. Reagent kit (50 times)
Substrate 1: 4 mg / ml rice glutelin solution 100 ml
Buffer 2: 0.1M lactate buffer (pH 4.0) 100 ml
Reaction stop solution 3: 0.4M TCA solution 300ml
Coloring reagent 4: ninhydrin solution 100 ml
Standard solution 5: 50 μg / ml arginine solution Reaction operation (i) 1 ml of substrate 1, 1 ml of buffer solution 2, and 0.2 ml of test enzyme solution 6 are mixed and reacted at 40 ° C. for 10 minutes to obtain reaction solution 7. The substrate blank solution 8 and the enzyme blank solution 9 are prepared by adding the same amount of distilled water as the substrate 1 and the test enzyme solution 6 respectively.
(Ii) 3 ml of the reaction stop solution 3 is mixed with the reaction solution 7, the substrate blank solution 8, and the enzyme blank solution 9 to stop the reaction. Then, it filtrates and the filtrate 10 is obtained.
(Iii) 1 ml of the coloring reagent 4 is added to 1 ml of the filtrate 10 and mixed, followed by reaction at 80 ° C. to 100 ° C. for 15 minutes to obtain a measurement sample 11.
(Iv) The measurement sample 11 is measured with a spectrophotometer at an absorbance of 570 nm.
(V) Separately, 1 ml of the standard solution 5 and 1 ml of the coloring reagent 4 are mixed, and a calibration curve is prepared by measuring the absorbance at 570 nm using a spectrophotometer. In addition, 1 ml of distilled water and the control solution 12 are mixed with 1 ml of the coloring reagent 4 as a background.

As described above, in measuring the proteolytic enzyme activity of sputum, the activity measurement can be easily and easily performed with high reproducibility by using a kit in which reagents used in advance are prepared.
It should be noted that the scale can be changed as long as those skilled in the art can technically use it. For example, in the case of a smaller scale, the absorbance may be measured with a plate reader using a 96-well plate or the like. The operation at each stage may be automatic measurement using an apparatus.

  As described above, the present invention has overcome the problems existing in the conventional method for measuring proteolytic enzyme activity. In other words, in the conventional method, the activity was not reflected in the amount of amino acid produced at the actual brewing site, but in the method for measuring the proteolytic enzyme activity of koji according to the present invention, the total protein of koji was obtained by using rice protein as a substrate. It has become possible to carry out a measurement that has a very high correlation with the amount of amino acid produced by the brew, which is important for the quality of the degrading enzyme activity. In particular, since the optimum pH for amylase and protease activity in koji is concentrated around 4, the actual sake is measured by measuring proteolytic enzyme activity between pH 3 and 4.5, which is the brewing condition of sake and shochu. It is possible to reflect the exact proteolytic enzyme activity when brewing or shochu.

  In addition, sake brewing starts fermentation as lactic acid acidity (pH 3) by adding lactic acid to prevent the growth of rotting lactic acid bacteria, and in the present invention, under such conditions, rice as a raw material, Rice glutelin is easily extracted from rice bran and white rice cake by dilute acid treatment. Unlike conventional methods, this can be performed without the need for a steaming step, so that the labor for substrate adjustment can be reduced.

  In addition, it has been clarified from correlation analysis that rice glutelin is mainly decomposed by rice bran enzyme into steamed rice protein, and other rice proteins that may inhibit proteolytic enzyme activity Rather than being mixed, the enzyme activity involved in amino acid production can be measured more accurately when the rice glutelin of the present invention is used as a main component and a substrate for proteolytic enzyme activity measurement.

  It is inferred from the genome analysis that Aspergillus oryzae has 134 proteolytic enzyme genes. Enzymatic degradation of rice protein in persimmon is considered to be due to the combined action of many enzymes. The present invention, which is possible, has a significant effect.

  In this embodiment, for sake brewing of sake and shochu, the case where rice protein is used as a substrate for measuring the proteolytic enzyme activity of koji is described. However, the raw material used for brewing is miso or soy sauce. For example, any food such as soybean, shochu, wheat, straw, corn, and molasses can be used, and an extract corresponding to each food can be used as a substrate for measuring the proteolytic enzyme activity of straw. Furthermore, as a substrate protein, glycinin which is a main component of soy protein can be used as long as it is a food derived from soybeans such as miso and soy sauce. Thus, the protein used as a substrate for measuring the proteolytic enzyme activity of sputum can be changed depending on the raw material used.

  It should be noted that the configuration, analysis, and measurement of the above embodiment are examples, and it is needless to say that they can be changed as appropriate without departing from the spirit of the present invention.

  Proteolytic enzyme activity measurement of the present invention using rice glutelin as a substrate shows the total activity of many proteolytic enzymes contained in sake lees and accurately measures the amount of rice protein degradation in sake and shochu brewing Can do. Therefore, it can be used for the measurement of proteolytic enzyme activity reflected in the amount of amino acid produced during the production of brewed products such as sake and shochu brewing. Great contributions are expected in the brewing field, such as improvement of rice, selection of glutinous rice varieties and breeding of new glutinous rice.

The figure which showed the two-dimensional electrophoresis of the whole rice protein. The figure which showed SDS-PAGE of the rice protein extracted from the white rice powder. The figure which showed the process flow of the proteolytic enzyme activity measuring method of this embodiment. The figure which showed the influence of pH which acts on the enzyme reaction of this embodiment. The figure which showed the enzyme degradation pattern of the rice protein of this embodiment. The comparison figure of the proteolytic enzyme activity measuring method of this embodiment and the National Tax Agency predetermined analysis method. The conceptual diagram of the proteolytic enzyme activity measuring kit of this embodiment.

Claims (5)

A substrate for measuring proteolytic enzyme activity, which is mainly composed of rice glutelin ,
The rice glutelin is
Add 0.05 to 1M lactic acid (pH 2.1 to 2.8) to rice powder, rice, rice bran, or white rice bran that is not defatted and not steamed, stir, let stand for 1 to 3 hours, and filter,
This rice protein is obtained by isoelectric precipitation at pH 6.4 to 10.0.
A substrate for measuring proteolytic enzyme activity. A method for producing a substrate for measuring proteolytic enzyme activity comprising rice glutelin as a main component,
The rice glutelin is
Add 0.05 to 1M lactic acid (pH 2.1 to 2.8) to rice powder, rice, rice bran, or white rice bran that is not defatted and not steamed, stir, let stand for 1 to 3 hours, and filter,
A method for producing a substrate for measuring proteolytic enzyme activity, which is a rice protein obtained by isoelectric focusing at pH 6.4 to 10.0. A substrate adjusting step for adjusting a substrate for measuring a protease activity according to claim 1 ;
酵素 Enzyme solution adjustment step to adjust enzyme solution,
A method for measuring proteolytic enzyme activity of salmon comprising an enzyme reaction step of reacting the substrate adjusted in the substrate adjustment step with the enzyme solution adjusted in the enzyme solution adjustment step. The method according to claim 3 , wherein the enzyme reaction step is performed at a pH of 3 to 5. Substrate adjusting means for adjusting a substrate for measuring proteolytic enzyme activity according to claim 1 ;
酵素 Enzyme solution adjusting means for adjusting the enzyme solution;
A kit for measuring proteolytic enzyme activity of salmon, comprising: an enzyme reaction means for reacting the substrate adjusted by the substrate adjustment means and the enzyme solution adjusted by the enzyme solution adjustment means.