Department of Biomolecular Health Sciences, Faculty of Veterinary Medicine, Utrecht University, Utrecht, Netherlands.
FrieslandCampina, Amersfoort, Netherlands.
J Nutr. 2021 Jun 1;151(6):1416-1425. doi: 10.1093/jn/nxab031.
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.
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.
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.
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).
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.
牛乳中含有细胞外囊泡（EVs），这些囊泡通过选择性地摄取生物活性分子来调节受体细胞的过程，从而充当细胞间通讯的介质。由于这些 EV 成分中的一些在进化上是保守的，因此商业牛奶中的 EV 有可能调节人类消费者的细胞过程。
由于商业牛奶经过工业加工，我们研究了其对分离牛奶 EV 数量和完整性及其生物活性成分的影响。为此，我们将从生牛乳中分离出的 EV 与不同类型的商业牛奶（包括巴氏杀菌奶、均质或非均质以及超高温处理（UHT）奶）中分离出的 EV 进行了比较。
通过差速离心分离 EV 与其他牛奶成分，然后通过密度梯度超速离心分离 EV。通过单颗粒高分辨率荧光流式细胞术比较不同牛奶类型的 EV，以确定 EV 的数量；通过冷冻电子显微镜观察 EV 的完整性和形态；通过 Western blot 分析研究 EV 相关蛋白载量；通过 RNA 分析评估总小 RNA 浓度和乳源性 EV 特定 microRNA 的表达。
在 UHT 牛奶中，我们无法检测到完整的 EV。有趣的是，尽管巴氏杀菌（无论均质与否）并不影响 EV 数量的平均值±标准差（与生牛乳相比，3.4×108±1.2×108-2.8×108±0.3×107），但它影响了 EV 的完整性和外观，改变了它们的蛋白质特征，并导致乳源性 EV 相关 RNA 的丢失（从生牛乳中的 40.2±3.4ng/μL 降至加工牛奶中的 17.7±5.4-23.3±10.0ng/μL，P<0.05）。
经过巴氏杀菌或 UHT 加热的商业牛奶分别含有较少或没有完整的 EV。尽管基于颗粒数，大多数 EV 似乎对巴氏杀菌具有抗性，但它们的完整性受到影响，其分子组成也发生了改变。因此，通过牛乳 EV 向人类消费者传递生物活性成分的可能性在经过热处理的商业牛乳中可能会降低或改变。
