Presentation Information
[P04-530]Retention Behavior of Milk Exosome-Like Vesicles and Casein Micelles in Anion-Exchange Chromatography
○Chika Adachi1, Noriko Yoshimoto1 (1. Yamaguchi University (Japan))
Keywords:
Milk Exosome-Like Vesicles,Casein Micelles,Anion-Exchange Chromatography
[Purpose]
Milk exosome-like vesicles (MEVs), nanosized vesicles of approximately 100 nm, are expected to serve as carriers for intercellular communication. However, their utilization is complicated by contamination with casein micelles, and separation methods capable of distinguishing subtle differences in surface charge and hydrophobicity are therefore required. In this study, we focused on Super Q-650M, an anion-exchange resin containing grafted polymer ligands (GPLs) in large pores, and investigated its retention behavior toward MEVs. Its separation performance was also compared with those of Q Sepharose FF/HP and Q Sepharose XL.
[Method]
Raw milk was defatted by centrifugation, treated with acetic acid, centrifuged to remove precipitates, and filtered through a 0.22 μm membrane. Linear salt-gradient elution experiments were carried out using pretreated raw milk samples and purified casein on columns packed with Q Sepharose FF/Q Sepharose HP (Column A), Q Sepharose XL (Column B), and Super Q-650M (Column C). The column effluent was fractionated into 96-well microplates, and phospholipid concentrations in each fraction were measured using the fluorescent dye FM4-64. The presence of MEVs was further confirmed using a Bovine Milk Exosome ELISA Kit.
[Results]
For Columns A and B, three peaks were observed: a non-retained peak at around 3 mL immediately after sample injection, a peak near 10 mL where the salt concentration began to increase, and a broad peak between 15 and 25 mL. Comparison with the elution profile of purified casein indicated that the peak eluting after 15 mL originated from residual casein that had not been completely removed during pretreatment.
For Column C, the pretreated raw milk sample showed a peak at around 18 mL after the start of the salt gradient. However, this peak appeared at nearly the same elution position as purified casein, indicating that separation of the remaining casein was difficult on Column C. Phospholipid measurements showed that phospholipids were present both in the peak eluted immediately after sample injection and in the peaks obtained during the gradient for all columns. In addition, the MEV detection kit confirmed the presence of MEVs in fractions collected at 2.5–2.7 mL for Column A and 19–21 mL for Column C.
[Consideration]
Separation of MEVs in raw milk was examined using three anion-exchange resins with different ligands and pore sizes. The results showed that MEVs were retained on the resin with larger pores. However, residual casein was also retained under these conditions, indicating that complete separation of MEVs from casein remains difficult.
[Conclusion]
Exosome-like particles in milk could be adsorbed and recovered by anion-exchange chromatography. Casein was also suggested to adsorb under similar conditions. Many casein micelle-like species did not adsorb on Sepharose-based resins with relatively small pores, whereas species interacting with larger pores were likely retained and co-eluted with the exosome-like particles. In addition, the adsorption capacity of these resins for MEVs remains unclear and should be investigated further.
Milk exosome-like vesicles (MEVs), nanosized vesicles of approximately 100 nm, are expected to serve as carriers for intercellular communication. However, their utilization is complicated by contamination with casein micelles, and separation methods capable of distinguishing subtle differences in surface charge and hydrophobicity are therefore required. In this study, we focused on Super Q-650M, an anion-exchange resin containing grafted polymer ligands (GPLs) in large pores, and investigated its retention behavior toward MEVs. Its separation performance was also compared with those of Q Sepharose FF/HP and Q Sepharose XL.
[Method]
Raw milk was defatted by centrifugation, treated with acetic acid, centrifuged to remove precipitates, and filtered through a 0.22 μm membrane. Linear salt-gradient elution experiments were carried out using pretreated raw milk samples and purified casein on columns packed with Q Sepharose FF/Q Sepharose HP (Column A), Q Sepharose XL (Column B), and Super Q-650M (Column C). The column effluent was fractionated into 96-well microplates, and phospholipid concentrations in each fraction were measured using the fluorescent dye FM4-64. The presence of MEVs was further confirmed using a Bovine Milk Exosome ELISA Kit.
[Results]
For Columns A and B, three peaks were observed: a non-retained peak at around 3 mL immediately after sample injection, a peak near 10 mL where the salt concentration began to increase, and a broad peak between 15 and 25 mL. Comparison with the elution profile of purified casein indicated that the peak eluting after 15 mL originated from residual casein that had not been completely removed during pretreatment.
For Column C, the pretreated raw milk sample showed a peak at around 18 mL after the start of the salt gradient. However, this peak appeared at nearly the same elution position as purified casein, indicating that separation of the remaining casein was difficult on Column C. Phospholipid measurements showed that phospholipids were present both in the peak eluted immediately after sample injection and in the peaks obtained during the gradient for all columns. In addition, the MEV detection kit confirmed the presence of MEVs in fractions collected at 2.5–2.7 mL for Column A and 19–21 mL for Column C.
[Consideration]
Separation of MEVs in raw milk was examined using three anion-exchange resins with different ligands and pore sizes. The results showed that MEVs were retained on the resin with larger pores. However, residual casein was also retained under these conditions, indicating that complete separation of MEVs from casein remains difficult.
[Conclusion]
Exosome-like particles in milk could be adsorbed and recovered by anion-exchange chromatography. Casein was also suggested to adsorb under similar conditions. Many casein micelle-like species did not adsorb on Sepharose-based resins with relatively small pores, whereas species interacting with larger pores were likely retained and co-eluted with the exosome-like particles. In addition, the adsorption capacity of these resins for MEVs remains unclear and should be investigated further.
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