Optimisation and comparison of orthogonal methods for separation and characterisation of extracellular vesicles to investigate how representative infant milk formula is of milk. (15th August 2021)

• Record Type: Journal Article
• Authors: Mukhopadhya, Anindya; Santoro, Jessie; Moran, Barry; Useckaite, Zivile; O'Driscoll, Lorraine
• Is Part Of: Food chemistry. Volume 353(2021)
• Publication Date: 2021-08-15
• Subjects: Extracellular vesicles (EVs) -- Milk -- Infant milk formula (IMF) -- Pre-treatment -- Developed methodologies -- EV-TRACKED; Food -- Analysis -- Periodicals; Food -- Composition -- Periodicals; 664
• DOI: 10.1016/j.foodchem.2021.129309
• Languages: English
• ISSNs: 0308-8146

Abstract

Highlights: Milk extracellular vesicles (EVs) contribute to cell growth and immunity. Identified means for milk and IMF EV separation, avoiding casein micelles. Acid treatment + gradient ultracentrifugation is essential to obtain enriched EVs. EVs exist in IMF, but are compromised compared to skim milk EVs. Both EV morphology and their cargo proteins are compromised in IMF. Abstract: Many infants are fed infant milk formula (IMF). However, IMF production from skim milk (SM) involves harsh treatment. So, we hypothesised that the quantity and/or quality of extracellular vesicles (EVs) in IMF may be reduced. Thus, firstly, we aimed to optimise separation of EVs from IMF and SM and, secondly, we aimed to compare the EV isolates from these two sources. Prior to EV isolation, abundant casein micelles of similar sizes to EVs were removed by treating milk samples with either acetic acid or hydrochloric acid. Samples progressed to differential ultracentrifugation (DUC) or gradient ultracentrifugation (GUC). EV characterisation included BCA, SDS-PAGE, nanoparticle tracking (NTA), electron microscopy (TEM), immunoblotting, and imaging flow cytometry (IFCM). Reduced EV concentrations were found in IMF. SM-derived EVs were intact, while IMF contained disrupted EV-like structures. EV biomarkers were more abundant with isolates from SM, indicating EV proteins in IMF are compromised. Altogether, a suitable method combining acid pre-treatment with GUC for EV separation from milk products was developed. EVs appear to be substantially compromised in IMF compared to SM.