Acidification effects on isolation of extracellular vesicles from bovine milk

Md Matiur Rahman; Kaori Shimizu; Marika Yamauchi; Hiroshi Takase; Shinya Ugawa; Ayaka Okada; Yasuo Inoshima

doi:10.1371/journal.pone.0222613

Acidification effects on isolation of extracellular vesicles from bovine milk

PLoS One. 2019 Sep 16;14(9):e0222613. doi: 10.1371/journal.pone.0222613. eCollection 2019.

Authors

Md Matiur Rahman^{1

2

3}, Kaori Shimizu², Marika Yamauchi², Hiroshi Takase⁴, Shinya Ugawa⁵, Ayaka Okada^{2

6}, Yasuo Inoshima^{1

2

6

7}

Affiliations

¹ The United Graduate School of Veterinary Sciences, Gifu University, Gifu, Gifu, Japan.
² Laboratory of Food and Environmental Hygiene, Cooperative Department of Veterinary Medicine, Gifu University, Gifu, Gifu, Japan.
³ Department of Medicine, Sylhet Agricultural University, Sylhet, Bangladesh.
⁴ Core Laboratory, Graduate School of Medical Sciences, Nagoya City University, Mizuho-cho, Mizuho-ku, Nagoya, Aichi, Japan.
⁵ Department of Anatomy and Neuroscience, Graduate School of Medical Sciences, Nagoya City University, Mizuho-cho, Mizuho-ku, Nagoya, Aichi, Japan.
⁶ Education and Research Center for Food Animal Health, Gifu University (GeFAH), Gifu, Gifu, Japan.
⁷ Joint Graduate School of Veterinary Sciences, Gifu University, Gifu, Gifu, Japan.

Abstract

Bovine milk extracellular vesicles (EVs) attract research interest as carriers of biologically active cargo including miRNA from donor to recipient cells to facilitate intercellular communication. Since toxicity of edible milk seems to be negligible, milk EVs are applicable to use for therapeutics in human medicine. Casein separation is an important step in obtaining pure EVs from milk, and recent studies reported that adding hydrochloric acid (HCl) and acetic acid (AA) to milk accelerates casein aggregation and precipitation to facilitate EV isolation and purification; however, the effects of acidification on EVs remain unclear. In this study, we evaluated the acidification effects on milk-derived EVs with that by standard ultracentrifugation (UC). We separated casein from milk by either UC method or treatment with HCl or AA, followed by evaluation of EVs in milk serum (whey) by transmission electron microcopy (TEM), spectrophotometry, and tunable resistive pulse sensing analysis to determine EVs morphology, protein concentration, and EVs size and concentration, respectively. Moreover, we used anti-CD9, -CD63, -CD81, -MFG-E8, -HSP70, and -Alix antibodies for the detection of EVs surface and internal marker proteins by western blot (WB). Morphological features of EVs were spherical shape and similar structure was observed in isolated EVs by TEM. However, some of the EVs isolated by HCl and AA had shown rough surface. Although protein concentration was higher in whey obtained by UC, EV concentration was significantly higher in whey following acid treatment. Moreover, although all of the targeted EVs-marker-proteins were detected by WB, HCl- or AA-treatments partially degraded CD9 and CD81. These findings indicated that acid treatment successfully separated casein from milk to allow efficient EV isolation and purification but resulted in partial degradation of EV-surface proteins. Our results suggest that following acid treatment, appropriate EV-surface-marker antibodies should be used for accurate assess the obtained EVs for downstream applications. This study describes the acidification effects on EVs isolated from bovine milk for the first time.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
Biomarkers / metabolism
Caseins / metabolism
Cattle
Extracellular Vesicles / metabolism*
Humans
MicroRNAs / metabolism
Milk / metabolism*
Ultracentrifugation / methods

Substances

Biomarkers
Caseins
MicroRNAs

Grants and funding

This study was partially supported by a research project grant for Improving Animal Disease Prevention Technologies to Combat Antimicrobial Resistance (2017–2021 FY to YI) from the Ministry of Agriculture, Forestry, and Fisheries of Japan. This study was also supported in part by the OGAWA Science and Technology Foundation and the Morinaga Foundation for Health and Nutrition. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.