Microstructural study of thermal denaturation and gelation of proteins using an Agilent 660 FTIR

Importance of the Topic

The microstructural changes associated with the thermal denaturation and gelation of β-lactoglobulin play a fundamental role in the functionality of whey-based food products. Understanding these processes enhances control over texture, stability, and quality in industrial formulations.

Objectives and Study Overview

This application note investigates the physicochemical behavior of β-lactoglobulin A over a temperature range of 40–92 °C using mid-infrared FTIR spectroscopy to elucidate unfolding and aggregation mechanisms during heat treatment.

Methodology and Used Instrumentation

A 5 % (w/v) solution of β-lactoglobulin B in deuterated phosphate buffer (pH 7) was analyzed in a temperature-controlled transmission cell. An Agilent Cary 660 FTIR spectrometer operated in rapid scan mode (5 kHz, 128 scans, 4000–800 cm⁻¹, 4 cm⁻¹ resolution) collected spectra while the sample was heated from 40 to 92 °C. Data acquisition and kinetic analysis were performed with Agilent Resolutions Pro software.

Results and Discussion

Deconvoluted FTIR spectra at 40 °C revealed seven amide I bands corresponding to distinct secondary structures: β-type (1691 cm⁻¹), β-sheet (1677, 1634 cm⁻¹), turns (1664 cm⁻¹), α-helix (1648 cm⁻¹), β-strand (1622 cm⁻¹), and side‐chain vibrations (1614 cm⁻¹). Above 76 °C, loss of bands at 1677, 1648, 1634, and 1614 cm⁻¹ indicated denaturation, while new bands at 1682 and 1617 cm⁻¹ signaled aggregation via intermolecular antiparallel β-sheet formation.

Practical Benefits and Applications

This approach provides real-time molecular-level insights into protein unfolding and gelation kinetics, facilitating optimized thermal processing in dairy and food industries and guiding formulation strategies for improved texture and stability.

Future Trends and Possibilities

Future work may integrate time-resolved 2D FTIR, coupling with rheology or calorimetry, and apply advanced chemometric analysis to further resolve transient intermediates and scale findings to complex food matrices.

Conclusion

The Agilent Cary 660 FTIR demonstrates high sensitivity for monitoring rapid structural transitions in β-lactoglobulin during heating, delivering valuable data to correlate secondary structure changes with gelation behavior in food applications.

Reference

• Ismail A, Kirkwood J. Microstructural study of thermal denaturation and gelation of proteins using an Agilent 660 FTIR. Agilent Technologies Application Note 5991-0019EN; March 26, 2012.