Acidification effects on isolation of extracellular vesicles from bovine milk
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 i...
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| description | 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. |
| doi_str_mv | 10.1371/journal.pone.0222613 |
| format | Article |
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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.</description><identifier>ISSN: 1932-6203</identifier><identifier>EISSN: 1932-6203</identifier><identifier>DOI: 10.1371/journal.pone.0222613</identifier><identifier>PMID: 31525238</identifier><language>eng</language><publisher>United States: Public Library of Science</publisher><subject>Acetic acid ; Acid deposition ; Acidification ; Animals ; Antibodies ; Biological activity ; Biology and Life Sciences ; Biomarkers ; Biomarkers - metabolism ; Cancer ; Casein ; Caseins - metabolism ; Cattle ; CD63 antigen ; CD81 antigen ; CD9 antigen ; Cell interactions ; Cell signaling ; Chemical properties ; Cow's milk ; Downstream effects ; Engineering and Technology ; Evaluation ; Extracellular vesicles ; Extracellular Vesicles - metabolism ; Food ; Heat shock proteins ; Hsp70 protein ; Humans ; Hydrochloric acid ; Hygiene ; Intercellular signalling ; Laboratories ; Lipids ; Medicine and Health Sciences ; Metabolism ; Methods ; MicroRNA ; MicroRNAs ; MicroRNAs - metabolism ; Milk ; Milk - metabolism ; Milk proteins ; miRNA ; Morphology ; Organic acids ; Protein purification ; Proteins ; Purification ; Reagents ; Research and Analysis Methods ; Spectrophotometry ; Therapeutics ; Toxicity ; Ultracentrifugation ; Ultracentrifugation - methods ; Vesicles ; Veterinary medicine ; Whey</subject><ispartof>PloS one, 2019-09, Vol.14 (9), p.e0222613</ispartof><rights>COPYRIGHT 2019 Public Library of Science</rights><rights>2019 Rahman et al. This is an open access article distributed under the terms of the Creative Commons Attribution License: http://creativecommons.org/licenses/by/4.0/ (the “License”), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.</rights><rights>2019 Rahman et al 2019 Rahman et al</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c758t-79de8e9b0cea69bbc65b20feb186e16fbc3f52ac060e62aa53b78eb0deb0d7423</citedby><cites>FETCH-LOGICAL-c758t-79de8e9b0cea69bbc65b20feb186e16fbc3f52ac060e62aa53b78eb0deb0d7423</cites><orcidid>0000-0002-1189-8392</orcidid></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC6746375/pdf/$$EPDF$$P50$$Gpubmedcentral$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC6746375/$$EHTML$$P50$$Gpubmedcentral$$Hfree_for_read</linktohtml><link.rule.ids>230,314,723,776,780,860,881,2096,2915,23845,27901,27902,53766,53768,79343,79344</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/31525238$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><contributor>Fan, Guo-Chang</contributor><creatorcontrib>Rahman, Md Matiur</creatorcontrib><creatorcontrib>Shimizu, Kaori</creatorcontrib><creatorcontrib>Yamauchi, Marika</creatorcontrib><creatorcontrib>Takase, Hiroshi</creatorcontrib><creatorcontrib>Ugawa, Shinya</creatorcontrib><creatorcontrib>Okada, Ayaka</creatorcontrib><creatorcontrib>Inoshima, Yasuo</creatorcontrib><title>Acidification effects on isolation of extracellular vesicles from bovine milk</title><title>PloS one</title><addtitle>PLoS One</addtitle><description>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.</description><subject>Acetic acid</subject><subject>Acid deposition</subject><subject>Acidification</subject><subject>Animals</subject><subject>Antibodies</subject><subject>Biological activity</subject><subject>Biology and Life Sciences</subject><subject>Biomarkers</subject><subject>Biomarkers - metabolism</subject><subject>Cancer</subject><subject>Casein</subject><subject>Caseins - metabolism</subject><subject>Cattle</subject><subject>CD63 antigen</subject><subject>CD81 antigen</subject><subject>CD9 antigen</subject><subject>Cell interactions</subject><subject>Cell signaling</subject><subject>Chemical properties</subject><subject>Cow's milk</subject><subject>Downstream effects</subject><subject>Engineering and Technology</subject><subject>Evaluation</subject><subject>Extracellular vesicles</subject><subject>Extracellular Vesicles - metabolism</subject><subject>Food</subject><subject>Heat shock proteins</subject><subject>Hsp70 protein</subject><subject>Humans</subject><subject>Hydrochloric acid</subject><subject>Hygiene</subject><subject>Intercellular signalling</subject><subject>Laboratories</subject><subject>Lipids</subject><subject>Medicine and Health Sciences</subject><subject>Metabolism</subject><subject>Methods</subject><subject>MicroRNA</subject><subject>MicroRNAs</subject><subject>MicroRNAs - metabolism</subject><subject>Milk</subject><subject>Milk - metabolism</subject><subject>Milk proteins</subject><subject>miRNA</subject><subject>Morphology</subject><subject>Organic acids</subject><subject>Protein purification</subject><subject>Proteins</subject><subject>Purification</subject><subject>Reagents</subject><subject>Research and Analysis Methods</subject><subject>Spectrophotometry</subject><subject>Therapeutics</subject><subject>Toxicity</subject><subject>Ultracentrifugation</subject><subject>Ultracentrifugation - methods</subject><subject>Vesicles</subject><subject>Veterinary medicine</subject><subject>Whey</subject><issn>1932-6203</issn><issn>1932-6203</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2019</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><sourceid>BENPR</sourceid><sourceid>DOA</sourceid><recordid>eNqNkmuL1DAUhoso7kX_gWhBEPwwYy5t0n4RhsXLwMqCt68hSU9mMqbNmLSD--9Nne4yBQUJIeH0OW-St2-WPcNoiSnHb3Z-CJ10y73vYIkIIQzTB9k5rilZMILow5P9WXYR4w6hklaMPc7OKC5JSWh1nn1aadtYY7Xsre9yMAZ0H_O0tdG7Y9GbHH71QWpwbnAy5AeIVjuIuQm-zZU_2A7y1rofT7JHRroIT6f1Mvv2_t3Xq4-L65sP66vV9ULzsuoXvG6gglohDZLVSmlWKoIMKFwxwMwoTU1JpEYMASNSllTxChRqxskLQi-zF0fdvfNRTE5EQUiNaVmUCCVifSQaL3diH2wrw63w0oo_BR82QoZ-fIVoeK1YU8mKgClAcVmogilFWNVg0LxIWm-n0wbVQqOhS2a4mej8S2e3YuMPgvGCUV4mgZeTQPA_B4j9P648URuZbmU740fPWxu1WJV1zUmBKU7U8i9UGg20VqcsGJvqs4bXs4bE9Ol3buQQo1h_-fz_7M33OfvqhN2CdP02RWYYIxPnYHEEdfAxBjD3zmEkxijfuSHGKIspyqnt-anr90132aW_AT7C8FY</recordid><startdate>20190916</startdate><enddate>20190916</enddate><creator>Rahman, Md Matiur</creator><creator>Shimizu, Kaori</creator><creator>Yamauchi, Marika</creator><creator>Takase, Hiroshi</creator><creator>Ugawa, Shinya</creator><creator>Okada, Ayaka</creator><creator>Inoshima, Yasuo</creator><general>Public Library of Science</general><general>Public Library of Science (PLoS)</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>IOV</scope><scope>ISR</scope><scope>3V.</scope><scope>7QG</scope><scope>7QL</scope><scope>7QO</scope><scope>7RV</scope><scope>7SN</scope><scope>7SS</scope><scope>7T5</scope><scope>7TG</scope><scope>7TM</scope><scope>7U9</scope><scope>7X2</scope><scope>7X7</scope><scope>7XB</scope><scope>88E</scope><scope>8AO</scope><scope>8C1</scope><scope>8FD</scope><scope>8FE</scope><scope>8FG</scope><scope>8FH</scope><scope>8FI</scope><scope>8FJ</scope><scope>8FK</scope><scope>ABJCF</scope><scope>ABUWG</scope><scope>AEUYN</scope><scope>AFKRA</scope><scope>ARAPS</scope><scope>ATCPS</scope><scope>AZQEC</scope><scope>BBNVY</scope><scope>BENPR</scope><scope>BGLVJ</scope><scope>BHPHI</scope><scope>C1K</scope><scope>CCPQU</scope><scope>D1I</scope><scope>DWQXO</scope><scope>FR3</scope><scope>FYUFA</scope><scope>GHDGH</scope><scope>GNUQQ</scope><scope>H94</scope><scope>HCIFZ</scope><scope>K9.</scope><scope>KB.</scope><scope>KB0</scope><scope>KL.</scope><scope>L6V</scope><scope>LK8</scope><scope>M0K</scope><scope>M0S</scope><scope>M1P</scope><scope>M7N</scope><scope>M7P</scope><scope>M7S</scope><scope>NAPCQ</scope><scope>P5Z</scope><scope>P62</scope><scope>P64</scope><scope>PATMY</scope><scope>PDBOC</scope><scope>PIMPY</scope><scope>PQEST</scope><scope>PQQKQ</scope><scope>PQUKI</scope><scope>PTHSS</scope><scope>PYCSY</scope><scope>RC3</scope><scope>5PM</scope><scope>DOA</scope><orcidid>https://orcid.org/0000-0002-1189-8392</orcidid></search><sort><creationdate>20190916</creationdate><title>Acidification effects on isolation of extracellular vesicles from bovine milk</title><author>Rahman, Md Matiur ; Shimizu, Kaori ; Yamauchi, Marika ; Takase, Hiroshi ; Ugawa, Shinya ; Okada, Ayaka ; Inoshima, Yasuo</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c758t-79de8e9b0cea69bbc65b20feb186e16fbc3f52ac060e62aa53b78eb0deb0d7423</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2019</creationdate><topic>Acetic acid</topic><topic>Acid deposition</topic><topic>Acidification</topic><topic>Animals</topic><topic>Antibodies</topic><topic>Biological activity</topic><topic>Biology and Life Sciences</topic><topic>Biomarkers</topic><topic>Biomarkers - 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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.</abstract><cop>United States</cop><pub>Public Library of Science</pub><pmid>31525238</pmid><doi>10.1371/journal.pone.0222613</doi><tpages>e0222613</tpages><orcidid>https://orcid.org/0000-0002-1189-8392</orcidid><oa>free_for_read</oa></addata></record> |
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| identifier | ISSN: 1932-6203 |
| ispartof | PloS one, 2019-09, Vol.14 (9), p.e0222613 |
| issn | 1932-6203 1932-6203 |
| language | eng |
| recordid | cdi_plos_journals_2291354500 |
| source | MEDLINE; DOAJ Directory of Open Access Journals; Elektronische Zeitschriftenbibliothek - Frei zugängliche E-Journals; PubMed Central; Free Full-Text Journals in Chemistry; Public Library of Science (PLoS) |
| subjects | Acetic acid Acid deposition Acidification Animals Antibodies Biological activity Biology and Life Sciences Biomarkers Biomarkers - metabolism Cancer Casein Caseins - metabolism Cattle CD63 antigen CD81 antigen CD9 antigen Cell interactions Cell signaling Chemical properties Cow's milk Downstream effects Engineering and Technology Evaluation Extracellular vesicles Extracellular Vesicles - metabolism Food Heat shock proteins Hsp70 protein Humans Hydrochloric acid Hygiene Intercellular signalling Laboratories Lipids Medicine and Health Sciences Metabolism Methods MicroRNA MicroRNAs MicroRNAs - metabolism Milk Milk - metabolism Milk proteins miRNA Morphology Organic acids Protein purification Proteins Purification Reagents Research and Analysis Methods Spectrophotometry Therapeutics Toxicity Ultracentrifugation Ultracentrifugation - methods Vesicles Veterinary medicine Whey |
| title | Acidification effects on isolation of extracellular vesicles from bovine milk |
| url | https://sfx.bib-bvb.de/sfx_tum?ctx_ver=Z39.88-2004&ctx_enc=info:ofi/enc:UTF-8&ctx_tim=2025-01-31T17%3A51%3A12IST&url_ver=Z39.88-2004&url_ctx_fmt=infofi/fmt:kev:mtx:ctx&rfr_id=info:sid/primo.exlibrisgroup.com:primo3-Article-gale_plos_&rft_val_fmt=info:ofi/fmt:kev:mtx:journal&rft.genre=article&rft.atitle=Acidification%20effects%20on%20isolation%20of%20extracellular%20vesicles%20from%20bovine%20milk&rft.jtitle=PloS%20one&rft.au=Rahman,%20Md%20Matiur&rft.date=2019-09-16&rft.volume=14&rft.issue=9&rft.spage=e0222613&rft.pages=e0222613-&rft.issn=1932-6203&rft.eissn=1932-6203&rft_id=info:doi/10.1371/journal.pone.0222613&rft_dat=%3Cgale_plos_%3EA599724131%3C/gale_plos_%3E%3Curl%3E%3C/url%3E&disable_directlink=true&sfx.directlink=off&sfx.report_link=0&rft_id=info:oai/&rft_pqid=2291354500&rft_id=info:pmid/31525238&rft_galeid=A599724131&rft_doaj_id=oai_doaj_org_article_d79b6d8a82ef4eb7a4b46bb268d1ec74&rfr_iscdi=true |