Determination of Amino Acidity in Japanese Sake Based on the Voltammetric Measurement of Surplus Acid by Quinone Reduction

Abstract

In this paper, we propose voltammetry for determining amino acidity in Japanese sake. The measurement principle of this voltammetric method is based on a concept of acid-base back titration and a decrease of cathodic prepeak current of 3,5-di-tert-butyl-1,2-benzoquinone (DBBQ) caused by surplus HCl, which is obtained by the neutralization between excess HCl and amino acids in Japanese sake. In the comparisons of amino acidities in Japanese sakes (n = 10) obtained by this voltammetric method and the official titration, called the ethanol additional method, the correlation coefficient (r) was 0.946, indicating that the amino acidities in Japanese sakes by both methods were essentially the same.

1. Introduction

Amino acids affect the flavor and taste of Japanese sake, and thus it is quite an important factor that determines the rich and/or umami tastes of Japanese sake.^1–3 In general, Japanese sakes with high amounts of amino acids are more full-bodied, have more umami, and have richer tastes, while Japanese sakes with low amounts of amino acids have lighter and cleaner tastes.^3,4 The total amino acid content in Japanese sake, called amino acidity, is expressed as the volume (mL) of 0.1 mol dm⁻³ (= M) NaOH required for the titration of formol nitrogen in 10 mL of Japanese sake,^5–7 where formol nitrogen means amino nitrogen determined by the formal titration. Amino acidity is one of the parameters for quality control of Japanese sake, and thus it is routinely measured in the brewing process of Japanese sake.

To determine amino acidity in Japanese sake, two kinds of neutralization titrations, called the formol titration and the ethanol additional method have been adopted as the official methods of the National Tax Agency JAPAN.^7–11 In formol titration, deleterious formalin is added in a titrand after the neutralization of acid components in Japanese sake, while the ethanol additional method uses ethanol instead of formalin. Although the use of formalin, which must be used in local exhaust ventilation systems, is avoided in the ethanol additional method, both titrations require a large sample volume, much time to conduct, and complicated procedures. Thus, a simple and specific analytical method such as a sensor for determining amino acidity is highly desired for the quality control of Japanese sake. An electroanalytical method, such as voltammetry, has a possibility to accomplish the above-mentioned purposes because it has attracted great attention for its relatively low cost, eco-friendly, and rapid measurements.^12,13 However, previous studies have shown that the main amino acids in Japanese sake are electrochemically inactive, and thus amino acid oxidase modified electrodes and derivatization reagents with electroactive groups are used for the electrochemical determination of amino acids.^14,15 To the best of our knowledge, electroanalytical methods including voltammetry have not been successfully developed for the determination of amino acidity in Japanese sake.

Previously, our group developed voltammetric sensing for determining acids based on the reduction of quinone such as 3,5-di-tert-butyl-1,2-benzoquinone (DBBQ).^16,17 The presence of a small amount of acid in an unbuffered protic solvent containing DBBQ was found to cause a new peak (termed a prepeak) at a more positive potential than the original cathodic peak of DBBQ itself on a voltammogram. The prepeak height of DBBQ increases with increasing amounts of acid in a manner proportional to the acid concentration, giving a basis for the voltammetric sensing of acids. Based on the findings of voltammetric sensing of acids,^16,17 we have introduced a voltammetric method for determining amino acids.¹⁸ As shown in Fig. S1 of the Supporting Information (SI), the voltammetric method was coupled with a concept of back neutralization titration and the voltammetric sensing of acids to determine amino acids. In this method, excess HCl as a strong acid titrant was added for neutralizing amino acids in a sample solution, and then the surplus acid was determined by measurement of the prepeak height of DBBQ. The concentration of amino acids in the sample solution was calculated from the results of the surplus acid determination. Recently, this voltammetric method has successfully been applied to the determination of free amino nitrogen in aged beef.¹⁹

In this study, based on our findings about the electrochemical determination of amino acids,¹⁸ we propose a voltammetric method as an electroanalytical method for determining amino acidity in Japanese sake. Moreover, because the miniaturization of the apparatus is easily achieved in voltammetry, a mobile sensor for personal use could be fabricated to select a favorite Japanese sake by measurement of amino acidity. As such, we have attempted to construct a prototype mobile device with a homemade potentiostat for the voltammogram measurement and it has been applied to determine amino acidity in Japanese sake.

2. Experimental

2.1 Chemicals and Japanese sakes

DBBQ (>98 %) was purchased from Tokyo Chemical Industry (Tokyo, Japan). Ethanol (99.5 %), NaCl (>99.5 %), concentrated HCl (analytical grade), and 0.1 M NaOH were purchased from FUJIFILM Wako Pure Chemical (Osaka, Japan). Pure water was obtained from an ultrapure water system (RFU666HA, ADVANTEC, Tokyo, Japan). Other chemicals and solvents were of reagent grade.

Samples of Japanese sakes were purchased from a supermarket in western Tokyo.

2.2 Voltammetry

2.2.1 Electrochemical cell and apparatus

A glassy carbon (3 mm diameter disk, BAS, Tokyo, Japan), an Ag/AgCl electrode, and a Pt wire were used as working, reference, and counter electrodes, respectively. These electrodes were inserted through the lid of a beaker-type cell with 6 mL of capacity.

A voltammogram was measured using an electrochemical measurement system (HZ-7000, Hokuto Denko, Tokyo, Japan) and/or a prototype mobile device. The prototype mobile device was constructed with a homemade potentiostat (13(W) × 9(D) × 6(H) cm, TOPPAN, Tokyo, Japan), a function generator (AWG-10K, ELMOS, Osaka, Japan), a mobile lithium-ion battery (CHE-059, TRA, Osaka, Japan), and a recorder (GL-100, GRAPHTEC, Kanagawa, Japan). A schematic diagram and a photograph of the prototype mobile device with a notebook computer are shown in Fig. S2 of the SI. These instruments were connected via multi-universal serial bus (USB) ports, and measured voltammograms from the recorder were transferred to the display on the monitor of the notebook computer. Scan and sampling rates in the electrochemical measurement system were set at 100 mV s⁻¹ and 10 points s⁻¹, respectively, while those in the prototype mobile device were set at 20 mV s⁻¹ and 2 points s⁻¹, respectively.

2.2.2 Preparation of test solution

Japanese sake (0.4 mL) was mixed with 35 mM HCl (0.6 mL) to neutralize the amino acids in the Japanese sake, and then 2 mL of an electrolyte cocktail (ethanol and water (9 : 1, v/v) mixture containing 16 mM DBBQ and 50 mM NaCl) were added to prepare the test solution. When the prepeak height of DBBQ was measured as a control, i.e. without Japanese sake, 0.4 mL of water were added instead of 0.4 mL of Japanese sake.

2.3 Titration based on the ethanol additional method

An automatic potentiometric titrator (AUT-701, DKK-TOA, Tokyo, Japan) was used to perform the titration for determining amino acidity in the Japanese sake, and the titration was processed according to the official method of the National Tax Agency JAPAN, called the ethanol additional method.^9–11 Firstly, 10 mL of Japanese sake were titrated with 0.1 M NaOH up to pH 8.2, and then 25 mL of ethanol were added to the titrand. Secondly, the titrand was subsequently titrated with 0.1 M NaOH up to pH 10.4 (endpoint). A volume (mL) of 0.1 M NaOH until the endpoint of the second titration, V, is defined as amino acidity in Japanese sake by the National Tax Agency JAPAN.^5–7 Amino acidity, V, is corrected by a factor of 0.1 M NaOH as necessary.

3. Results and Discussion

3.1 Measurement of prepeak height of DBBQ caused by HCl

Voltammograms of DBBQ without and with 7 mM HCl were measured in a water-ethanol mixture (6 : 4, v/v) containing 10.6 mM DBBQ and 33.3 mM NaCl using the electrochemical measurement system. In the absence of HCl, a cathodic peak of DBBQ was observed at −0.52 V vs. Ag/AgCl as shown in Fig. 1A (curve a). In the presence of HCl, a cathodic prepeak of DBBQ was observed at −0.24 V vs. Ag/AgCl as shown in Fig. 1A (curve b). The prepeak heights of DBBQ (i_Ha) were found to be linearly related to the concentration of HCl in the test solution, ranging from 70.0 µM to 7.00 mM (r = 0.998) as shown in Fig. 2. The repeatability for the measurements of the i_Ha was examined by repetitive measurements. The relative standard deviations (RSD, n = 6) of the i_Ha at 1 mM, 3 mM, and 6 mM HCl were 0.7 %, 0.9 %, and 1.2 %, respectively, indicating the voltammetric method had sufficient repeatability for determining HCl concentration in the test solution.

Figure 1.

Voltammograms of DBBQ in the presence of 7 mM HCl (A) before and (B) after neutralization with Japanese sake measured using the electrochemical measurement system. The voltammogram of DBBQ without HCl is shown as a curve (a), i.e. blank, and that with HCl is shown as a curve (b) in each figure. The i_Ha means the current of prepeak height of DBBQ with 7 mM HCl, and the i_Hb means the current of prepeak height of DBBQ after Japanese sake is neutralized with 7 mM HCl. The scan rate was set at 100 mV s⁻¹.

Figure 2.

Calibration curve of HCl. The i_Ha means the current of prepeak height of DBBQ caused by HCl in a test solution.

3.2 Voltammetric method for determining amino acidity in Japanese sake

In our previous study,¹⁸ we measured a voltammogram of DBBQ in the presence of HCl after glutamine was neutralized with excess HCl. The prepeak height of DBBQ was shown to decrease with the addition of glutamine, and the decrease in prepeak height of DBBQ was proportional to the glutamine concentration. Further, the slopes of the calibration curves for the diacid bases (e.g. lysine, etc.) were approximately twice compared with those for the monoacid bases (e.g. glycine, etc.). Thus, we show that an equivalent concentration of amino acids as bases was determined by the voltammetric method. Moreover, the voltammetric method based on the reduction of DBBQ was not interfered with sugar, ascorbic acid, polyphenol, tocopherols, caffeine, or inorganic compounds.¹⁹ In addition, the decrease of the prepeak height of DBBQ was not interfered with weak acids such as succinic acid because the prepeak of DBBQ caused by the weak acids was separately observed at a more negative potential than that by HCl. Figure S3 of the SI shows a voltammogram of DBBQ in the presence of 2.0 mM succinic acid and 5.0 mM HCl. Although the ill-defined prepeak of DBBQ caused by the succinic acid appeared at −0.32 V vs. Ag/AgCl, the prepeak heights of DBBQ caused by the HCl were almost the same without and with succinic acid. Because the appearance of a prepeak potential of quinone is dependent on the acidity of compounds,²⁰ it is expected that a proton of succinic acid is not donated to the reduction of DBBQ at a potential where the prepeak of DBBQ caused by HCl appears.

Similar voltammetry as shown in Fig. 1A (curve b) was performed after 0.4 mL of Japanese sake were neutralized with 0.6 mL of 35 mM HCl. The prepeak of DBBQ caused by the unneutralized HCl in the test solution was observed on the voltammogram, and the prepeak height of DBBQ in Fig. 1B (curve b) decreased in comparison with that in Fig. 1A (curve b). In other words, the decrease in the prepeak height of DBBQ in Fig. 1B (curve b) can be ascribed to the decreasing concentration of HCl by the neutralization with amino acids in the Japanese sake. Thus, an equivalent concentration of neutralized amino acids in Japanese sake, C_AA (mM), can be determined from the measurements of the prepeak heights of DBBQ caused by HCl and a concept of acid-base back neutralization titration as follows:   

\begin{equation} C_{\text{AA (mM)}} = 7_{\text{(mM)}} \times \frac{i_{\text{Ha}} - i_{\text{Hb}}}{i_{\text{Ha}}} \times \frac{3_{\text{(mL)}}}{0.4_{\text{(mL)}}} \end{equation} (1)

where 7, 3, and 0.4 indicate the concentration of HCl (mM) in the test solution as a control, the volume (mL) of the test solution in this voltammetric method, and the volume (mL) of Japanese sake used as a sample, respectively. In addition, the i_Ha means the current of the prepeak height of DBBQ with 7 mM HCl, while i_Hb means the current of the prepeak height of DBBQ after Japanese sake is neutralized with 7 mM HCl.

Considering the definition of amino acidity in Japanese sake, amino acidity, V, is expressed as follows:   

\begin{equation} V = \frac{C_{\text{AA (mM)}} \times 10_{\text{(mL)}}}{100_{\text{(mM)}}} = C_{\text{AA}} \times 0.1 \end{equation} (2)

where 100 and 10 indicate the concentration of NaOH (mM) and sample volume (mL) of Japanese sake used for titration, respectively.

From the prepeak heights obtained from the voltammograms in Fig. 1, amino acidity in Japanese sake (#1) was determined as 1.9. Meanwhile, the amino acidity in Japanese sake (#1) determined by the official titration was 1.8. Moreover, the determination of amino acidity in nine kinds of commercial Japanese sakes (#2–10) was performed by the voltammetric method using the electrochemical measurement system, and these results were compared with those by the official titration as shown in Table 1. The regression expression for the correlative relationship between the amino acidity obtained by the official titration and the voltammetric method was y = 0.972x − 0.055 with a correlation coefficient (r) of 0.946, where y and x are the amino acidity obtained by the official titration and the voltammetric method, respectively. Each amino acidity obtained by the official titration and the voltammetric method was found to be in good agreement. From these results, it is shown that the voltammetric method provided an accurate electroanalytical measurement for determining amino acidity in Japanese sake. According to the sample preparation described in the Experimental section, the voltammetric method can be used to determine amino acidity in Japanese sake ranging from 0.0525 to 5.25 because the linear range of HCl in the test solution was from 70.0 µM to 7.00 mM (Fig. 2).

Table 1. Determination of amino acidity in Japanese sake samples by the voltammetric method and the official titration.

Japanese sake		Amino acidity
#	Origin (Prefecture)	Voltammetric methoda	Official titrationb
1	Akita	1.9	1.8
2	Ishikawa	1.6	1.5
3	Gifu	1.3	1.2
4	Kanagawa	1.8	1.7
5	Miyagi	1.7	1.7
6	Niigata	1.7	1.6
7	Niigata	1.6	1.5
8	Tokyo	1.8	1.6
9	Nagano	1.4	1.2
10	Niigata	1.4	1.4

Note: ^a, Voltammograms were measured using the electrochemical measurement system.

^b, The ethanol additional method was performed as the official titration.

The voltammetric method and the official titration determine amino acids as bases and acids, respectively. Thus, basic amino acids (arginine, lysine, etc.) and acidic amino acids (aspartic acid, glutamic acid, etc.) cause positive and negative errors, respectively, when the results of amino acidity determined by the voltammetric method are compared with those of the official titration. In the total amino acid content in Japanese sakes, basic amino acids and acidic amino acids are present 9–17 % and 10–14 %, respectively.¹ It is possible that differences due to basic and acidic amino acid compositions in Japanese sakes produce positive or negative errors of amino acidity determined by the voltammetric method.

3.3 Prototype mobile device for determining amino acidity in Japanese sake

Using a homemade potentiostat, a prototype mobile device was developed for determining amino acidity in Japanese sake. We evaluated the accuracy and precision of the prototype mobile device by determining amino acidity. In the measurement of a voltammogram using our prototype mobile device, because the highest sampling rate is 2 points s⁻¹ on the recorder, the scan rate was set at 20 mV s⁻¹ to obtain a voltammogram with high resolution. A voltammogram of DBBQ in the presence of 7 mM HCl is shown in Fig. S4A of the SI, and one after neutralization with Japanese sake is shown in Fig. S4B of the SI. It was shown that sufficient voltammograms were obtained to determine amino acidity in Japanese sake using the prototype mobile device. The amino acidities in Japanese sakes (#1 and #2) with repeatability are shown in Table 2. The amino acidities in Japanese sakes by the voltammetric method using the prototype mobile device were approximately the same compared with that by the official titration and the voltammetric method using the electrochemical measurement system. The repeatability of the voltammetric method was found to be inferior when compared with that of the official titration, however, the voltammetric method has sufficient repeatability for determining amino acidity in Japanese sake. It was also found that the prototype mobile device could provide an accurate and precise determination of amino acidity in Japanese sake. For one assay, the official titration requires 10 mL of Japanese sake and 10 min of measurement time, while the voltammetric method requires 0.4 mL of Japanese sake and less than 1 min of measurement time. As such, the voltammetric method is useful to reduce measurement time and save sample volume for determining amino acidity in Japanese sake. Because a mobile lithium-ion battery supplies the electric power to work the prototype mobile device, it could be to manufacture a mobile sensor for personal use to help select a favorite Japanese sake by the measurement of amino acidity.

Table 2. Comparison of amino acidity determined in Japanese sake by the voltammetric method and the official titration.

Japanese sake		Amino acidity (RSD [%, n = 3])
#	Origin (Prefecture)	Voltammetric methoda	Voltammetric methodb	Official titrationc
1	Akita	1.9 (2.2)	1.9 (3.1)	1.8 (0.1)
2	Ishikawa	1.7 (1.8)	1.6 (2.4)	1.5 (0.5)

Note: ^a, Voltammograms were measured using the prototype mobile device.

^b, Voltammograms were measured using the electrochemical measurement system.

^c, The ethanol additional method was performed as the official titration.

4. Conclusion

In this paper, we have demonstrated amino acidity in Japanese sake was determined by voltammetry based on a concept of acid-base back titration and surplus acid measurement by a reduction prepeak of DBBQ. The results obtained by the voltammetric method were in good agreement with those by the official titration. Therefore, we have shown that the voltammetric method, with facility and economy, is useful in routine work for the quality control of Japanese sake. Moreover, we have achieved the development of a prototype mobile device to measure voltammograms, and it was applied to determine amino acidity in Japanese sake. Our report is the first one regarding the voltammetric determination of amino acidity in Japanese sake. In the end, we show the voltammetric method is both practical and valuable in determining amino acidity in Japanese sake.

Acknowledgments

The authors wish to thank Mr. Kenichiro Hakuta, Mr. Shinya Kusunoki, Mrs. Akiko Nagai, and Mr. Yuichiro Abe, TOPPAN, Japan, for their technical support to help prepare the prototype mobile device. This work was supported in part by the Japan Society for the Promotion of Science (JSPS) KAKENHI Grant Number JP22K05176.

Data Availability Statement

The data that support the findings of this study are openly available under the terms of the designated Creative Commons License in J-STAGE Data at https://doi.org/10.50892/data.electrochemistry.21268677.

CRediT Authorship Contribution Statement

Akira Kotani: Conceptualization (Lead), Formal analysis (Lead), Funding acquisition (Lead), Investigation (Lead), Methodology (Lead), Project administration (Lead), Visualization (Lead), Writing – original draft (Lead), Writing – review & editing (Lead)

Jumpei Watanabe: Data curation (Lead), Formal analysis (Lead), Investigation (Lead), Methodology (Lead), Validation (Lead)

Koichi Machida: Resources (Supporting), Supervision (Supporting), Writing – review & editing (Supporting)

Kazuhiro Yamamoto: Resources (Supporting), Supervision (Supporting), Writing – review & editing (Supporting)

Hideki Hakamata: Project administration (Lead), Resources (Lead), Supervision (Lead), Writing – original draft (Lead), Writing – review & editing (Lead)

Conflict of Interest

The authors declare no conflict of interest in the manuscript.

Funding

Japan Society for the Promotion of Science: JP22K05176

Footnotes

A. Kotani: ECSJ Active Member

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