Analysis of Chiral Amino Acids within Fermented Beverages Utilizing a Column Switching System

Significance of Chiral Amino Acid Analysis in Fermented Beverages

Fermented foods and beverages contain a variety of D-amino acids that influence taste, nutritional value and bioactivity. While L-amino acids prevail in fresh produce, fermentation processes generate detectable levels of D-isomers. Accurate profiling of these chiral amino acids supports quality control, product development and sensory optimization in the food and beverage industry.

Study Objectives and Overview

This study demonstrates a liquid chromatography–mass spectrometry (LC-MS) method using high-pressure column switching for simultaneous separation of D- and L-amino acids. The goal was to apply this approach to black vinegar and yogurt beverages, assess D/L ratios of key amino acids and compare chiral profiles across different fermented samples.

Methodology and Instrumentation

Sample pretreatment involved liquid–liquid extraction with water, methanol and chloroform, followed by centrifugation steps at 10 000 rpm and 4 °C. A small aliquot of the final supernatant was mixed with mobile phase prior to injection.

• Liquid Chromatograph: Shimadzu system with high-pressure column switching valves (FCV) alternating CROWNPAK CR-I(+) and CR-I(–) chiral columns (3 × 150 mm, 5 µm).
• Mobile Phase: acetonitrile/ethanol/water/TFA = 80/15/5/0.5; flow rate 0.6 mL/min; oven temperature 20 °C.
• Mass Spectrometer: ESI in positive ion mode; probe voltage +4.0 kV; nebulizing gas 3 L/min; drying gas 15 L/min; DL temperature 250 °C; interface temperature 250 °C; block heater 300 °C.

Main Results and Discussion

In black vinegar samples, D-alanine, D-arginine, D-asparagine and D-aspartic acid were detected at D/L ratios ranging from 0.6 % to 20.5 %, with vinegar B exhibiting the highest D-alanine content (~20 %). Yogurt beverages contained a broader spectrum of D-amino acids and notably high D-glutamic acid, exceeding L-glutamic acid by more than 40-fold. Comparative analysis highlighted that yogurt matrices support greater generation or retention of D-isomers such as D-alanine and D-lysine relative to black vinegar.

Benefits and Practical Applications of the Method

Column-switching LC-MS enables rapid, sensitive and selective chiral separation without derivatization. The approach is well suited for routine quality control in food manufacturing, sensory research linking chiral profiles to taste properties and nutritional studies on bioactive D-amino acids.

Future Trends and Potential Applications

Advances may include coupling with high-resolution MS for structural confirmation, automation of sample preparation workflows and application to diverse fermented products such as cheese, soy sauce and probiotic beverages. Correlating D-amino acid patterns with flavor, microbial activity and health effects will further expand the utility of chiral amino acid analysis.

Conclusion

The high-pressure column switching LC-MS technique offers a robust, high-throughput platform for comprehensive chiral amino acid profiling in complex fermented matrices. Its sensitivity and selectivity make it a valuable tool for food quality assurance, research and product innovation.

Reference

• Nakano Y., Konya Y., Taniguchi M., Fukusaki E., Journal of Bioscience and Bioengineering, 123, 134–138 (2016)