Analysis of Organic Acids Including Levulinic Acid in Soy Sauce

March 23, 2021

Introduction

The methods of making soy sauce are roughly classified into an authentically-brewed method and a mixed fermented method. The authentically-brewed method is a basic method using fermented soybeans. In the mixed fermented method, amino acid liquid generated by decomposition of plant protein such as soybean by acid or enzyme is mixed to Moromi and is fermented.

Levulinic acid  is in the amino acid liquid used in mixed fermented method, which was created by decomposition of soy bean. The trace levulinic acid remaining distinguishs the method of making soy sauce.

Organic acid is known as a substance of “Umami” and sour taste, and the functional properties such as antioxidative and antibacterial effect are attracting attention. Levulinic acid is classified in keto acid, and is one of the organic acids.

In this article, levulinic acid and organic acids are measured by a post column HPLC method. BTB is used as a pH indicator, and detection is performed at 445 nm by using UV-vis absorbance detector. The sample is authentically-brewed soy sauce with levulinic acid added.

LC-4000 HPLC system

Experimental

Experimental Condition
Column:   Shodex RSpak KC-G (6.0 mmI.D. x 50 mmL) + KC-811 x 2 (8.0 mmI.D. x 300 mmL  x 2)
Eluent:   2 mM Perchloric acid
Eluent flow rate:   1.0 mL/min
Reagent:  0.2 mM BTB + 15 mM Disodium hydrogen phosphate
Reagent flow rate:   1.5 mL/min
Column temp.:   60ºC
Detector: UV-vis absorbance detector (Wavelength: 445 nm)
Injection volume:   50 µL
Standard:   13 organic acids, 5 mmol/L each
Sample:  Soy sauce

Schematic diagram


1:Eluent, 2:Degassing unit, 3:Eluent pump, 4:Autosampler, 5:Gard column (RSpak KC-G), 6, 7:Column (RSpak KC-811), 8:Column oven, 9:   Reagent, 10:Reagent pump, 11:Organic acid reaction unit, 12:UV/VIS detector, 13:Chromatography data system

Keywords

Levulinic acid, Organic acid, Soy sauce, pH indicator, BTB, UV-vis absorbance detector

Results

Figure 1 shows the chromatogram of the organic acids mixture standard solution including levulinic acid.

Figure 1. Chromatogram of organic acids mixture standard solution including levulinic acid (5 mmol/L each, 1: Citric acid, 2: Malic acid, 3: Succinic acid, 4: Lactic acid, 5: Formic acid, 6: Acetic acid, 7: Levulinic acid, 8: Pyroglutamic acid, 9: Propionic acid, 10: Isobutyric acid, 11: n-Butyric acid, 12: Isovaleric acid, 13: n-Valeric acid)

Figure 2 shows the chromatogram of soy sauce added with levulinic acid (0.5 mmol/L). The soy sauce is diluted with ultrapure water by ten times.

Figure 2. Chromatogram of soy sauce added with levulinic acid (1: Citric acid, 3: Succinic acid, 4: Lactic acid, 5: Formic acid, 6: Acetic acid, 7: Levulinic acid, 8: Pyroglutamic acid)

Pretreatment: Soy sauce is diluted with ultrapure water by ten times, then it is filtered by 0.45 µm membrane filter. And then levulinic acid is added.

About the Author

Chromatography Group