Microstructure of milk gel and cheese curd observed using cryo scanning electron microscopy and confocal microscopy

Cryo scanning electron microscopy (cryo SEM) and confocal laser scanning microscopy (CLSM) were used to visualise changes in the microstructure of milk, rennet-induced gel and curd during the manufacture of Cheddar cheese. Our results show that cryo preservation did not alter the microstructure of the sample when it was fixed by rapid freezing in slush liquid nitrogen due to the formation of amorphous ice. Artefacts such as the formation of ice crystals could be observed in samples when immersed directly into liquid nitrogen (−196 °C) at atmospheric pressure. These ice crystals changed the shape of sample pores increasing their size to >20 μm. The etching time, thickness of gold coating, accelerating voltage and type of detector used for cryo SEM observation were varied in order to minimise the formation of such artefacts and optimise conditions for imaging. Chains and clusters of casein micelles and fat globules were best observed in the gel and the cooked curd when the samples were freeze fractured and etched for 30 min, coated with a mixture of gold and palladium alloy approximately 6 nm thick at −140 °C and observed using a backscattered electron detector at 15 kV. The structure of the gel, curd and cheese was also observed using CLSM. Spherical fat globules were mostly present in the serum pores of the gel prepared from unhomogenised milk but were found embedded in the aggregated chains of the casein network within the gel prepared from homogenised milk when observed using CLSM. The porosity measurements obtained using cryo SEM were similar to those obtained using CLSM. These two complementary techniques can potentially be used to assist studies for the control of cheese texture and functionality.