Characterization of fructans in fermentation products with GPC/SEC

Importance of the Topic

Biorefineries aim to valorize all biomass fractions to improve sustainability and economic efficiency. Fructans, plant-derived polymers of fructose, represent a valuable resource for food, feed, and specialty chemical markets. Accurate characterization and isolation of these compounds from fermentation broths enable downstream processing, quality control, and development of continuous purification strategies.

Study Objectives and Overview

This study focused on (1) profiling the molecular weight distribution of fructans and other sugars in a fermentation sample derived from Silphium perfoliatum, and (2) scaling the analytical protocol to a semi-preparative format for fractionation of oligomers from low-molecular-weight sugars. Additionally, the feasibility of implementing a simulated moving bed (SMB) process for continuous separation was evaluated.

Methodology and Sample Preparation

The frozen fermentation sample was thawed, centrifuged at 10 000 g for 30 minutes, and filtered through a 0.45 µm regenerated cellulose membrane. Analytical measurements used a 1:20 dilution, while semi-preparative injections employed the undiluted broth.

A universal SEC calibration was established with ten PEG standards (Mp 106–21 160 Da) at 0.1% (w/v) in water, applying Mark–Houwink parameters K=10.4 and α=0.57. Analytical separations were conducted on a 300×8 mm AppliChrom ABOA-SuperOH-P column (7 µm, 250 Å) at 25 °C, 1 ml/min flow, and refractive index detection. Semi-preparative scale-up maintained column length and particle size while increasing diameter from 8 mm to 20 mm and flow from 1 ml/min to 7 ml/min. Fractions of 7.5 ml were collected for subsequent analysis.

Used Instrumentation

• Isocratic pump AZURA P6.1L
• Autosampler AZURA AS 6.1L
• Refractive index detector AZURA RID 2.1L and high-flow RID 2.1L
• Thermostat AZURA CT 2.1
• AppliChrom ABOA-SuperOH-P SEC columns (300×8 mm and 300×20 mm)
• Fraction collector Foxy R1
• ClarityChrom software with SEC/GPC extension
• KNAUER ScaleUp Converter

Main Results and Discussion

The calibration achieved a fifth-order polynomial fit with R²=0.99978, enabling reliable molecular weight estimation. Analytical SEC resolved two distinct peaks: fructan oligomers (average Mw 20 247 Da, ~112 fructose units) and low-molecular-weight sugars (monomers to trimers). Semi-preparative separations maintained baseline resolution under scaled conditions. Fractions F6 and F7 were confirmed to contain predominantly fructan oligomers, with F6 showing the highest purity.

Benefits and Practical Applications

• Rapid qualitative and semi-quantitative profiling of fructan polymers in complex bioprocess samples.
• Preparative isolation of oligomer fractions suitable for functional testing or further refinement.
• Method adaptability to different polymers and process streams by adjusting calibration parameters.
• Foundation for continuous SMB operations to enhance throughput and reduce solvent consumption.

Future Trends and Applications

Advances may include integration of multi-angle light scattering detectors for absolute molar mass determination, implementation of continuous counter-current chromatography to increase productivity, and adaptation of the workflow to other carbohydrate polymers. Further development of high-throughput online SEC-RID platforms could support real-time process monitoring in industrial biorefineries.

Conclusion

This work demonstrates that size exclusion chromatography with refractive index detection provides a robust tool for both qualitative characterization and preparative fractionation of fructans from biotechnological feedstocks. The approach offers a clear path toward scalable purification and supports the development of continuous separation processes.

References

1. Mayr J., von Gehren P., Gansberger M. et al. The potential of the energy crop Silphium perfoliatum L. Proceedings of the 23rd European Biomass Conference and Exhibition, Vienna, Austria (2015).
2. Pollock C.J., Chatterton N.J. Fructans. In: Stumpf P.K., Conn E.E. (eds) The Biochemistry of Plants, Vol. 14, pp. 109–140. Academic Press (2012).
3. KNAUER ScaleUp Converter. KNAUER Scientific Devices GmbH (accessed 2020-01-12).