Beyond Single-Stain Controls in Plant-Based Cheese Microstructure Analysis: A tri-phase approach for validating...

Area of Science:

• Food Science
• Analytical Chemistry
• Microscopy

Background:

• Plant-based cheese development necessitates precise microstructural analysis.
• Existing methods struggle with simultaneous visualization of key components like starch, protein, and fat.

Purpose of the Study:

• To establish a validated confocal laser scanning microscopy protocol for simultaneous visualization of protein, fat, and starch in plant-based cheese.
• To systematically evaluate fluorescent staining methods for accuracy and specificity.

Main Methods:

• A tri-phase experimental approach was used, starting with single-component gels and progressing to complex ternary systems.
• Staining specificity of fluorescent probes, including Calcofluor White (CFW) and Fluorescent Brightener 28, was rigorously validated.
• Confocal laser scanning microscopy was employed for simultaneous three-component imaging.

Main Results:

• Commercial Calcofluor White (CFW) preparations showed cross-reactivity with proteins, hindering accurate starch visualization.
• Fluorescent Brightener 28 was identified as a superior stain, enabling selective starch labeling without protein interference.
• A robust protocol for simultaneous protein, fat, and starch visualization was successfully established.

Conclusions:

• The developed methodology provides accurate microstructural analysis for plant-based cheese systems.
• This advancement is crucial for optimizing product development in the growing plant-based food market.