Fermentation Processes Monitoring Using a Nexera™ Dual Injection System

Significance of the Topic

Fermentation underpins both traditional food production and modern bioprocessing in industries ranging from dairy to pharmaceuticals. Monitoring key metabolites such as organic acids and sugars is essential for optimizing yields, ensuring product consistency, and maintaining quality control. High‐performance liquid chromatography (HPLC) is a gold‐standard technique for this purpose, but conventional setups often require separate instruments for different analyte classes, which increases complexity and reduces throughput.

Objectives and Study Overview

This application note demonstrates the use of a dual injection HPLC system to perform simultaneous analysis of organic acids and sugars in a single run. Using yogurt fermentation as a model, the study aims to showcase how integrating two analytical channels on one platform can streamline process monitoring and data management.

Methodology and Instrumentation

• Instrumentation: Shimadzu Nexera HPLC system with dual injection autosampler (SIL‐40), two CTO‐40S column ovens, conductivity and refractive index detectors, and an integrated data acquisition module.
• Columns: Shim-pack Fast-OA ion-exclusion column for organic acids; Shim-pack SCR-101C ligand-exchange column for sugars; each paired with appropriate guard columns.
• Mobile Phases: 5.0 mmol/L p-toluenesulfonic acid buffer with Bis-Tris/EDTA for organic acids; water for sugar analysis.
• Operating Conditions: Flow rates of 0.8 mL/min (acids) and 1.0 mL/min (sugars); column temperatures of 40 °C and 80 °C; 10 µL injection volume for both channels.
• Sample Preparation: Yogurt fermented at 40 °C, sampled at defined intervals, acidified, extracted with chloroform, centrifuged, filtered through 0.45 µm membrane, and diluted 1:10 prior to injection.

Main Results and Discussion

• Linearity: All organic acids (citric, malic, lactic, formic, acetic) and sugars (lactose, glucose, mannose, fructose) exhibited calibration curves with R² ≥ 0.9998 over 10–2000 mg/L ranges.
• Reproducibility: Retention time and peak area %RSD were below 1% across six replicate injections, confirming method precision.
• Carryover: Post‐run rinsing achieved carryover rates below 0.01% for all analytes, ensuring reliable quantitation.
• Recovery: Spike-recovery in yogurt matrices ranged from 94.6% to 107.7%, demonstrating effective sample cleanup and minimal matrix interference.
• Fermentation Profiling: Time-course analysis revealed progressive lactose depletion and lactic acid accumulation, illustrating the system’s capability for dynamic process monitoring.

Benefits and Practical Applications

The dual injection approach consolidates multiple analyses into a single HPLC platform, reducing laboratory footprint and simplifying workflow. Integrated data management improves traceability, while simultaneous detection accelerates feedback for quality control and process optimization in fermentation-based industries.

Future Trends and Potential Applications

Extending dual injection HPLC to other metabolite classes and coupling with at-line or online sampling could enable real-time process analytical technology (PAT). This methodology is adaptable to diverse bioprocesses, including biofuel production, wastewater treatment, and pharmaceutical fermentations.

Conclusion

The Nexera dual injection HPLC system effectively enables concurrent quantitation of organic acids and sugars with high precision, low carryover, and robust sample recoveries. Its integrated design offers a powerful tool for comprehensive fermentation monitoring, supporting enhanced productivity and quality assurance.