TY - JOUR
T1 - Regular Industrial Processing of Bovine Milk Impacts the Integrity and Molecular Composition of Extracellular Vesicles
AU - Kleinjan, Marije
AU - van Herwijnen, Martijn Jc
AU - Libregts, Sten Fwm
AU - van Neerven, Rj Joost
AU - Feitsma, Anouk L
AU - Wauben, Marca Hm
N1 - Funding Information:
Ger Arkesteijn (Department of Biomolecular Health Sciences, Utrecht University) is acknowledged for expert support of the flow cytometric EV analysis,Tom AP Driedonks (Department of Biomolecular Health Sciences, Utrecht University) for excellent support in RNA analysis, and Willie Geerts (Bijvoet Center for Biomolecular Research, Utrecht University) for excellent technical support in the cryo-EM analysis. The authors' responsibilities were as follows-MHMW, MK, RJJvN, and ALF: conceived and designed the study; MK, SFWML, and MJCvH: performed the experiments and/or analyzed the data; MJCvH, MK,MHMW, ALF, and RJJvN: wrote and revised the manuscript; and all authors: contributed to final editing of the manuscript and read and approved the final manuscript
Publisher Copyright:
© 2021 The Author(s).
PY - 2021/6/1
Y1 - 2021/6/1
N2 - BACKGROUND: Bovine milk contains extracellular vesicles (EVs), which act as mediators of intercellular communication by regulating the recipients' cellular processes via their selectively incorporated bioactive molecules. Because some of these EV components are evolutionarily conserved, EVs present in commercial milk might have the potential to regulate cellular processes in human consumers. OBJECTIVES: Because commercial milk is subjected to industrial processing, we investigated its effect on the number and integrity of isolated milk EVs and their bioactive components. For this, we compared EVs isolated from raw bovine milk with EVs isolated from different types of commercial milk, including pasteurized milk, either homogenized or not, and ultra heat treated (UHT) milk. METHODS: EVs were separated from other milk components by differential centrifugation, followed by density gradient ultracentrifugation. EVs from different milk types were compared by single-particle high-resolution fluorescence-based flow cytometry to determine EV numbers, Cryo-electron microscopy to visualize EV integrity and morphology, western blot analysis to investigate EV-associated protein cargo, and RNA analysis to assess total small RNA concentration and milk-EV-specific microRNA expression. RESULTS: In UHT milk, we could not detect intact EVs. Interestingly, although pasteurization (irrespective of homogenization) did not affect mean ± SD EV numbers (3.4 × 108 ± 1.2 × 108-2.8 × 108 ± 0.3 × 107 compared with 3.1 × 108 ± 1.2 × 108 in raw milk), it affected EV integrity and appearance, altered their protein signature, and resulted in a loss of milk-EV-associated RNAs (from 40.2 ± 3.4 ng/μL in raw milk to 17.7 ± 5.4-23.3 ± 10.0 mg/μL in processed milk, P < 0.05). CONCLUSIONS: Commercial milk, that has been heated by either pasteurization or UHT, contains fewer or no intact EVs, respectively. Although most EVs seemed resistant to pasteurization based on particle numbers, their integrity was affected and their molecular composition was altered. Thus, the possible transfer of bioactive components via bovine milk EVs to human consumers is likely diminished or altered in heat-treated commercial milk.
AB - BACKGROUND: Bovine milk contains extracellular vesicles (EVs), which act as mediators of intercellular communication by regulating the recipients' cellular processes via their selectively incorporated bioactive molecules. Because some of these EV components are evolutionarily conserved, EVs present in commercial milk might have the potential to regulate cellular processes in human consumers. OBJECTIVES: Because commercial milk is subjected to industrial processing, we investigated its effect on the number and integrity of isolated milk EVs and their bioactive components. For this, we compared EVs isolated from raw bovine milk with EVs isolated from different types of commercial milk, including pasteurized milk, either homogenized or not, and ultra heat treated (UHT) milk. METHODS: EVs were separated from other milk components by differential centrifugation, followed by density gradient ultracentrifugation. EVs from different milk types were compared by single-particle high-resolution fluorescence-based flow cytometry to determine EV numbers, Cryo-electron microscopy to visualize EV integrity and morphology, western blot analysis to investigate EV-associated protein cargo, and RNA analysis to assess total small RNA concentration and milk-EV-specific microRNA expression. RESULTS: In UHT milk, we could not detect intact EVs. Interestingly, although pasteurization (irrespective of homogenization) did not affect mean ± SD EV numbers (3.4 × 108 ± 1.2 × 108-2.8 × 108 ± 0.3 × 107 compared with 3.1 × 108 ± 1.2 × 108 in raw milk), it affected EV integrity and appearance, altered their protein signature, and resulted in a loss of milk-EV-associated RNAs (from 40.2 ± 3.4 ng/μL in raw milk to 17.7 ± 5.4-23.3 ± 10.0 mg/μL in processed milk, P < 0.05). CONCLUSIONS: Commercial milk, that has been heated by either pasteurization or UHT, contains fewer or no intact EVs, respectively. Although most EVs seemed resistant to pasteurization based on particle numbers, their integrity was affected and their molecular composition was altered. Thus, the possible transfer of bioactive components via bovine milk EVs to human consumers is likely diminished or altered in heat-treated commercial milk.
KW - bovine milk
KW - commercial milk
KW - cow milk
KW - exosomes
KW - extracellular vesicles
KW - microRNA
KW - microvesicles
KW - pasteurization
KW - raw milk
KW - ultra heat treated
UR - http://www.scopus.com/inward/record.url?scp=85107293372&partnerID=8YFLogxK
U2 - 10.1093/jn/nxab031
DO - 10.1093/jn/nxab031
M3 - Article
C2 - 33768229
SN - 0022-3166
VL - 151
SP - 1416
EP - 1425
JO - Journal of Nutrition
JF - Journal of Nutrition
IS - 6
ER -