Effect of different pre-treatment methods on size determination of casein nanoparticles by dynamic light scattering

Abstract: The size and size distribution of casein nanoparticles plays an important role in the functionality and stability of milk. Dynamic light scattering is an effective method of measuring the size and size distribution of casein nanoparticles. But as biomacromolecules, casein nanoparticles are quite sensitive to their microenvironment changes. And therefore, the physicochemical properties of casein nanoparticles could be easily altered in different treatments, resulting in the incorrect size information determined by dynamic light scattering. The object of the present research was to analyze the effect of pre-treatment methods on the size and size distribution of casein nanoparticles determined by dynamic light scattering. The results suggested that when water and calcium imidazole buffer were used as the diluents, the counting rate of the samples was less than 7 ×103 counts/s, indicating there were no particles that could influence the size determination of casein nanoparticles. However, it was also found when water and calcium imidazole buffer were used as the dilution of casein nanoparticles, a significant decrease in the counting rate could be observed. This indicated both water and calcium imidazole buffer could easily alter the microenvironments of casein micelle dispersions, resulting in the partially dissociation of nanoparticles. And thus, the water and calcium imidazole buffer were not suitable for the dilution of casein nanoparticles. When simulated milk ultrafiltrate and milk ultrafiltrate were used as the diluents, it was found that there were no significant changes in the counting rate of the casein micelle dispersions. This indicated the microenvironment of both simulated milk ultrafiltrate and milk ultrafiltrate (10 kDa) was quite similar to that of milk, and thus the casein nanoparticles could not dissociate in both simulated milk ultrafiltrate and milk ultrafiltrate. However, extra particles occurred in both simulated milk ultrafiltrate and milk ultrafiltrate 24 h after their preparation, and therefore they were not suitable for the size measurement of casein nanoparticles under such the situation. Furthermore, it could be observed that the temperature of the diluents could significantly affect the measurement accuracy: when the samples (25℃) were diluted with 4℃ diluents, the counting rate could reduce by 16%; when the samples (4℃) were diluted with 25℃ diluents, the counting rate could increase by 10%. Based on the conclusion obtained above, it was suggested that the best pre-treatment methods for the size and size distribution of casein nanoparticles by dynamic light scattering were: the temperatures of the samples diluted with simulated milk ultrafiltrate or milk ultrafiltrate (10 kDa) should be similar with the milk samples. Results indicated the size distribution of casein nanoparticles obtained by dynamic light scattering was quite similar with that observed by the cryo-transmission electron microscopy, indicating the pre-treatment methods used could not affect the structural properties of casein nanoparticles in their true microenvironments, and thus permitted us to obtain reliable information of size and size distribution.