JPH0558137B2 - - Google Patents

Description

[Detailed description of the invention]

INDUSTRIAL APPLICATION FIELD The present invention relates to a total amino acid analysis method that is effectively adopted for quantifying amino acids contained in alcohols such as Japanese sake. Conventional technology and its problems Changes in Japan's industrial structure have affected the way of life in rural areas, and the number of sake brewers, who were engaged in sake brewing during the agricultural off-season, is rapidly decreasing, causing serious problems in the sake brewing industry. It's becoming a problem. Under these circumstances, in the industry, alcohol, total amino acids, sugar,
Streamline by managing total acid, specific gravity, etc. with a simple meter,
There has been a movement to try to improve the constitution. Among these measurement items, the National Tax Agency's prescribed analysis method has been used to measure total amino acids in alcoholic beverages. According to this analysis method prescribed by the National Tax Agency, amino acids exist as zwitterions in water, but
Titration is not possible due to its weak dissociation. However, in a neutral aqueous solution, the dissociation equilibrium shifts to the acid side in the presence of formalin, and the amino group forms a stable compound, so if phenolphthalein is used as an indicator, it becomes possible to titrate with an alkali. That is, take 10 ml of a sample, add 2 to 3 drops of phenolphthalein indicator, neutralize with N/10 sodium hydroxide solution, add 5 ml of neutral formalin solution, and remove the liberated acid with N/10 sodium hydroxide solution. Titrate with the solution until it turns pale pink. This titrated ml number is defined as a, and a method is adopted in which it is expressed as the amino acid content using the following formula. Amino acid content = a × F (F: N/10 factor of sodium hydroxide solution) In this case, the method shown above can be automated, but the equipment is large-scale, so the sake brewing industry has many small and medium-sized enterprises. However, there was a need for a method for analyzing all amino acids that would be simpler, more automated, and more abbreviated. SUMMARY OF THE INVENTION In view of the above circumstances, the present inventors conducted intensive studies on a method for easily measuring the amount of amino acids contained in sake, etc., and as a result, selected an alkaline phosphate buffer as the buffer solution. By adding formalin to this to create a flow system of formalin-phosphate buffer, and injecting the sample containing amino acids into this, the mixed solution of formalin-buffer-sample is adjusted according to the amount of amino acids in the sample. We have discovered that the PH decreases by a certain degree, and that by measuring and detecting the degree of this PH decrease with a PH measuring electrode, it is possible to reliably quantify the amino acid concentration, and have come to form the present invention. be. In other words, in addition to the manual titration method described above, there are other methods for analyzing amino acids, such as liquid chromatography, enzyme electrode method, and the colorimetric ninhydrin method. Believing that this method would be more acceptable to the sake brewing industry, we tried to automate the analysis method prescribed by the National Tax Agency as faithfully as possible, and considered changing the method of detecting PH changes from the indicator method to the PH measurement electrode method, taking into account the simplicity of measurement. the result,
We found that alkaline phosphate buffer is optimal as described below, and created a flow system by adding formalin to this phosphate buffer, and created a flow system in which formalin-phosphate buffer continuously flows. When sake is injected into the sake, the amino acids contained in the sake are converted to organic acids, and the PH of the buffer solution shifts to the acid side. This PH shift corresponds to the total amount of amino acids contained in the sake, so the PH changes. By detecting the degree of deviation, the total amount of amino acids can be measured, and in this case
Since the pH measurement electrode is extremely stable in alkaline phosphate buffer, the degree of pH deviation can be reliably detected using the pH measurement glass electrode. Therefore, this method allows reliable, quick and easy quantification of all amino acids. This is what we found out that it can be done. Hereinafter, the present invention will be explained in more detail with reference to the drawings. Structure of the Invention The total amino acid analysis method of the present invention involves introducing a sample containing amino acids into a continuous flow system in which formalin and an alkaline phosphate buffer are mixed, and the formalin-alkaline phosphate buffer produced at this time. The degree of PH reduction in a mixed solution of buffer solution and sample is detected with a PH measurement electrode, and the amino acid concentration in the sample is determined from this PH reduction degree. Here, the method of the present invention will be explained with reference to the principle diagram shown in Fig. 1. In the flow system shown in Fig. 1, pump P is operated and an acid-base buffer (HA) is
-A ^- ) and the sample acid (HB) on the other hand at flow rates of V and V, respectively, the electrode potential _E1 of the PH sensor S determined by the flow of the buffer solution is expressed by the following formula: shown. E ₁ = E° + 59logK _a,HA +59logC° _HA /C° _A (mV) where K _a,HA is the acid dissociation constant, C° _HA and C° _A are the initial concentrations of HA and A ^- , respectively. In this case, when the ratio of C _HA /C _A changes by mixing the buffer solution and the acid HB, the potential E ₂ becomes E ₂ = E° + 59logK _a,HA + 59log {(C° _HA V+xv)/(C°
_A V−xv)}. Note that x indicates the concentration of acid. v/V=1
The potential change ΔE due to sample mixing is ΔE=59log [{1+(x/C° _HA )}/{1−(x/C°
_A ^- ) ^} ] ^Therefore , ^the acid _{concentration} x ^is _expressed by ^the following formula ^/59 can be obtained. When HB is a weak acid, x is equal to the initial concentration of HB, C° _HB , but when it is a weak acid with an acid dissociation constant of K _{a, HB} , it becomes as follows. That is, the acid dissociation constants of HA and HB are K _A and K _B
Then, when acid HB is added as a sample to a buffer solution of HA and A ^- , the reaction equation can be expressed as A ^- + HBHA + B ^- , and if the equilibrium constant is K, then K = (C° _HA +x) x / (C ° _A −x) (C° _HB −x)=K _B
/K _A , x = 1/2 (K-1) [{K (C° _A + C° _HB ) + C° _HB }
−√{(° _A +° _HB )+° _HA } ² −4(−1)
It can be expressed as ° _A ° _HB . However, K=K _B /K _A ,
When C° _HA = C° _A , the above ΔE is plotted against the ratio of C° _HB /C° _HA as shown in Figure 2, and C° _HB /
In the range of C° _HA from 0 to 0.5, the potential change has a linear relationship with the added sample concentration C° _HB . From FIG. 2, it can be seen that if the acid dissociation constant of the sample is 10 ³ or more than that of HA in the buffer, the curves will be the same. In the present invention, the stability of the electrode potential in the buffer is utilized, so the selection of the buffer is important. Therefore, ammonium chloride/ammonium water,
As a result of adding glycine as an amino acid to phosphate-sodium hydrogen/sodium hydroxide and sodium carbonate/sodium hydrogen carbonate buffer solutions, we investigated the PH changes. Although no PH change was observed with the sodium/sodium hydrogen carbonate buffer, the alkaline phosphate buffer mentioned above has a large PH change and is linear, as shown in Section 3, making it ideal for this method. This is what I discovered. In addition, the phosphate buffer (0.1M-NaHPO ₄ -0.05MNaOH system, pK _a =
12.3) PH change in 5 ml of glycine, 5 ml of 18% formaldehyde (neutral), 10 ml of 0.1M-NaHPO ₄ ,
These are the results of measuring PH after adding 10 ml of 0.05M-NaOH. In other words, the pK _a of the acid produced by reacting with formalin
is about 6 to 7, so if we consider the theoretical curve shown in Figure 2, if we use a buffer solution that is a certain distance from the pK _a , we can measure with the same sensitivity regardless of the type of amino acid. _{A 4} ^2- -PO ₄ ^3- based phosphate buffer is optimal, and for these reasons, an alkaline phosphate buffer is selected as the buffer in the present invention. Therefore, as is clear from what has been said above,
In the present invention, by injecting a sample containing amino acids into a formalin-alkaline phosphate buffer flow system, the pH decreases in proportion to the amount of amino acids, and the degree of this decrease in pH is measured using a pH measurement electrode. This method was completed based on the knowledge that the amount of amino acids can be determined by the following methods. When carrying out the present invention, the flow rate of formalin-alkaline phosphate buffer should be 0.5 to 3 ml/min, particularly 1 to 2 ml/min. The mixing ratio of formalin and phosphate buffer varies depending on the amount of amino acids in the sample, etc., but the mixing ratio of formalin and phosphate buffer is such that the concentration of formalin is about 100 times the concentration of amino acids. It is preferable to determine the mixing ratio of the phosphate buffer. In addition, as the alkaline phosphate buffer, HPO ₄ ^2-
-PO ₄ ^3- based compounds can be suitably used. Although the amount of sample introduced into the formalin-alkaline phosphate buffer is not necessarily limited,
The thickness is preferably 2 to 500μ. Further, the electrodes used for PH measurement and the measurement method thereof can be conventional methods, and the amount of amino acids can be determined from the PH measurement results by methods such as using a calibration curve. FIG. 4 shows an example of the apparatus used for carrying out the present invention, in which 1 is a phosphate buffer tank, 2 is a formalin tank, 3 is a pure water tank, 4 is a pump, 5 is a sample injector, and 6 is a Flow cell, 7 is a pH measurement electrode, 8 is a reference electrode, 9 is an ion meter, 10 is a recorder,
Reference numeral 11 denotes a drain tank, in which a pump 4 is operated to flow phosphate buffer, formalin, and pure water at a predetermined flow rate, and a sample containing an amino acid is injected into the pure water from a sample injector 5. The mixed liquid is introduced into the flow cell 6, and the pH of the mixed liquid is measured and recorded on the recorder 10. Note that in the above example, the sample was injected into the formalin channel, and then the formalin channel and the phosphate buffer channel were merged, but after the formalin channel and the phosphate buffer channel were merged, , the sample may be injected into this merging channel. The results of quantifying all amino acids in Japanese sake using the apparatus shown in FIG. 4 are shown in Examples below. Example The total amino acids in Japanese sake were analyzed by the following method. Phosphate buffer (0.1M NaHPO ₄ −
0.05M NaOH system pK _a = 12.3), 18% formalin solution, and pure water were set at a flow rate of 0.5 ml/min and flowed through the flow cell to stabilize the electrode potential. Once the readings are stable, create a calibration curve. To create a calibration curve, use L-valine 1×10 ^-2 , 2×10 ^-2 , 4
Prepare a ×10 ^-2 M solution and inject 130μ of each using a syringe. The injected L-valine is oxidized by formalin as shown in the following formula, H ₂ NC ₄ H ₈ COOH + nHCHO → HOH ₂ CHNC ₄ H ₈ COOH or (HOH ₂ C) ₂ N
C ₄ H ₈ COOH Converted to organic acid and mixed in phosphate buffer. An acid-base reaction takes place in the buffer, and this pH
Changes in pH are detected using a glass electrode for PH measurement and a reference electrode inserted into the flow cell. The peak height from the standard is measured and a calibration curve as shown in FIG. 5 is created. In addition, since glycine, leucine, and L-valine have the same dissociation constant in formalin as the amino acids used for creating the calibration curve, the same calibration curve was obtained regardless of which amino acid was used.
In addition, the peak detection method may be either a method of measuring the peak height or a method of determining the potential difference. Next, inject the sample, convert the various amino acids contained in the sake into organic acids using the same procedure as above, calculate the peak height from the PH change of the buffer solution, and use the previously determined calibration curve to convert the various amino acids contained in the sake into organic acids. Determine the total amount of amino acids. Note that the amount of amino acids may be calculated by directly recording the concentration on a printer using a data processing unit. Table 1 shows the results of purchasing five types of commercially available Japanese sake from different manufacturers, determining the total amino acid content in the sake using this method, and comparing it with the analysis method prescribed by the National Tax Agency.

[Table] From the results in Table 1, this method has a correlation coefficient with that of the conventional method.
0.939, showing good agreement, indicating that it is possible to quantify all amino acids in sake using this method. Furthermore, FIG. 6 shows the peak reproducibility and response time when samples from manufacturers B and C were analyzed three times each. Peak reproducibility is 2.6 and CV, respectively.
2.8%, and the reaction time for one measurement was 30 seconds in both cases B and C. Furthermore, Figure 7 shows that when glycine was used as a sample in the titration of amino acids according to the analysis method prescribed by the National Tax Agency (2 × 10 ^-2 M/
(using 10ml of glycine and 10ml of 6M formaldehyde)
The titration curve was shown. By comparing the chromatogram in FIG. 6 with the titration curve in FIG. 7, it is clear that the method of the present invention is highly practical. Next, from the above results, it was found that the concentration of total amino acids in sake is about 1.50 × 10 ^-2 to 2.15 × 10 ^-2 M/, but it is difficult to follow the concentration of total amino acids in this method outside of this concentration range. The properties were investigated using sake from Manufacturer B. That is, the amino acid concentration is 2.05×
To prepare a solution more concentrated than 10 ^-2 M, add L-valine to adjust the amount of amino acids in the sake.
When preparing a solution with an amino acid concentration of 2.05×10 ⁻² M/more diluted, it was diluted with pure water. Six samples were prepared using this method, and the total amino acid content was determined using this method and the analysis method prescribed by the National Tax Agency in the same manner as described above. The results are shown in FIG. In the results of this study, the regression equation of this method and the analysis method specified by the National Tax Agency shows a good correlation with y=0.997x−0.019 and a correlation coefficient of 0.997, and this method follows the changes in the concentration of all amino acids in sake. I found out that there is a sex. Above, we have described examples of applying this method to quantify all amino acids in Japanese sake, but we have also described other examples in which this method is applied to quantify all amino acids in synthetic sake, mirin, beer, and wine, and process control of all amino acids in fermentation production processes. This law was valid until then. In addition, in the example, the sample was introduced into the measurement system using a sample injector, but instead of the injector, a metering pump was used to continuously feed a fixed amount of the sample. A similar result was obtained. Effects of the Invention In the amino acid analysis method according to the present invention, a sample containing amino acids is injected into a flow system in which formalin-alkaline phosphate buffer solution flows continuously, and a mixed solution of formalin-buffer solution-sample is generated. The method is characterized in that the degree of PH reduction in the sample is detected with a PH measurement electrode, and the amino acid concentration in the sample is determined from the degree of PH reduction, and has the following characteristics. (1) Since it is based on a continuously flowing PH buffer, the baseline is extremely stable, and the reproducibility of the peak height of minute PH changes caused by acid-base reactions is extremely good. (2) There is no need to draw a titration curve; all you need to do is measure the peak height, so quantitative determination is very quick. (3) The relationship between total amino acids and peak height is not logarithmic, which is unique to the electrode method, but is proportional, making it easy to calculate the concentration. (4) The system consists of a pump, injector, detector,
All you need is a receiver, recorder, etc., and since it is simple, it is easy to automate. (5) Since PH measurement electrodes for flow analysis are used to detect PH changes, selectivity is high, and sample stains, coloring, etc. have almost no effect on quantitative determination. (6) The sample used for analysis only needs to be about 100μ, which is an extremely small amount. (7) Analytical capacity is fast, 120 samples per hour. (8) To quantify all amino acids, it is enough to simply inject the sample, and no special expertise is required, contributing to labor savings.

[Brief explanation of the drawing]

Figure 1 is a schematic diagram explaining the principle of the present invention, Figure 2 is a schematic diagram explaining the principle of the invention.
The figure shows the theoretical curve of potential change in acid-base titration using a PH electrode. Figure 3 shows the potential change of phosphate buffer when glycine is added.
A graph showing PH changes, Fig. 4 is a schematic diagram showing an example of an apparatus used for implementing the present invention, Fig. 5 is a graph showing an example of a calibration curve when L-valine is used, and Fig. 6 is a schematic diagram showing an example of a calibration curve using L-valine. Chromatogram showing the reproducibility of peak height when the method is applied to the analysis of total amino acids in sake. Figure 7 is a titration curve of amino acids in formalin based on the conventional analysis method specified by the National Tax Agency. Figure 8 is a titration curve of amino acids in formalin. It is a graph showing the correlation between the method of the present invention and the conventional analysis method prescribed by the National Tax Agency. 1... Phosphate buffer tank, 2... Formaldehyde tank, 3... Pure water tank, 4... Pump, 5... Sample introducer, 6... Flow cell, 7... Electrode for PH measurement, 8... Comparison Electrode, 9...Ion meter.

Claims (1)

[Claims] 1. A sample containing amino acids is introduced into a continuous flow system in which formalin and alkaline phosphate buffer are mixed, and the degree of PH drop in the mixed solution of formalin, alkaline phosphate buffer, and sample that occurs at this time is measured. A total amino acid analysis method that detects with a PH measurement electrode and determines the amino acid concentration in a sample from the degree of PH decrease.