Department of Nutrition and Health Sciences, University of Nebraska-Lincoln, Lincoln 68583.
Department of Biochemistry, University of Nebraska-Lincoln, Lincoln 68588.
J Dairy Sci. 2021 Sep;104(9):9478-9493. doi: 10.3168/jds.2021-20300. Epub 2021 Jul 1.
Extracellular vesicles (EV) in milk, particularly exosomes, have attracted considerable attention as bioactive food compounds and for their use in drug delivery. The utility of small EV in milk (sMEV) as an animal feed additive and in drug delivery would be enhanced by cost-effective large-scale protocols for the enrichment of sMEV from byproducts in dairy plants. Here, we tested the hypothesis that sMEV may be enriched from byproducts of cheesemaking by tangential flow filtration (EV-FF) and that the sMEV have properties similar to sMEV prepared by ultracentrifugation (sMEV-UC). Three fractions of EV were purified from the whey fraction of cottage cheese making by using EV-FF that passed through a membrane with a 50-kDa cutoff (50 penetrate; 50P), and subfractions of 50P that were retained (100 retentate; 100R) or passed through (100 penetrate; 100P) a membrane with a 100-kDa cutoff; sMEV-UC controls were prepared by serial ultracentrifugation. The abundance of sMEV (<200 nm) was less than 0.3% in EV-FF compared with sMEV-UC (10/mL of milk). Despite the low EV count, the protein content (mg/mL) of 100R (63 ± 0.02; ± standard deviation) was higher than that of 50P (0.75 ± 0.10), 100P (0.65 ± 0.40), and sMEV-UC (27 ± 0.02). There were 17, 14, 35, and 75 distinct proteins detected by nontargeted mass spectrometry analysis in 50P, 100R, 100P, and sMEV-UC, respectively. Exosome markers CD9, CD63, CD81, HSP-70, PDCD6IP, and TSG101 were detected in control sMEV-UC but not in EV-FF by using targeted mass spectrometry and immunoblot analyses. Negative exosome markers, APOB, β-integrin, and histone H3 were below the limit of detection in EV-FF and control sMEV-UC analyzed by immunoblotting. The abundance of the major milk fat globule protein butyrophilin showed the following pattern: 100R ≫ 100P = 50P > sMEV-UC. More than 100 mature microRNA were detected in sMEV-UC by using sequencing analysis, compared with 36 to 60 microRNA in EV-FF. Only 100R and sMEV-UC yielded mRNA in quantities and qualities sufficient for sequencing analysis; an average of 276,000 and 838,000 reads were mapped to approximately 14,600 and 18,500 genes in 100R and sMEV-UC, respectively. In principal component analysis, microRNA, mRNA, and protein in EV-FF preparations clustered separately from control sMEV-UC. We conclude that under the conditions used here, flow filtration yields a heterogeneous population of milk EV.
乳细胞外囊泡（EV），特别是外泌体，作为生物活性食品化合物吸引了相当大的关注，并被用于药物递送。通过成本效益高的大规模方案从乳制品工厂的副产品中富集小 EV（sMEV），可以增强小 EV 在牛奶中的应用（sMEV）作为动物饲料添加剂和药物递送的效用。在这里，我们测试了以下假设：sMEV 可能通过切向流过滤（EV-FF）从干酪制作的副产物中富集，并且 sMEV 具有与通过超速离心（sMEV-UC）制备的 sMEV 相似的特性。使用具有 50 kDa 截止值的膜（50 穿透；50P）从乳清部分中通过 EV-FF 纯化了三种 EV 级分，并且截留（100 保留物；100R）或通过具有 100 kDa 截止值的膜（100 穿透；100P）的 50P 亚级分；sMEV-UC 对照通过连续超速离心制备。与 sMEV-UC（10/mL 牛奶）相比，EV-FF 中 sMEV（<200nm）的丰度小于 0.3%。尽管 EV 计数较低，但 100R（63 ± 0.02；±标准差）的蛋白质含量（mg/mL）高于 50P（0.75 ± 0.10）、100P（0.65 ± 0.40）和 sMEV-UC（27 ± 0.02）。通过非靶向质谱分析分别在 50P、100R、100P 和 sMEV-UC 中检测到 17、14、35 和 75 种独特的蛋白质。通过靶向质谱分析和免疫印迹分析，在对照 sMEV-UC 中检测到外泌体标志物 CD9、CD63、CD81、HSP-70、PDCD6IP 和 TSG101，但在 EV-FF 中未检测到。在 EV-FF 和对照 sMEV-UC 中通过免疫印迹分析，阴性外泌体标志物 APOB、β-整联蛋白和组蛋白 H3 的含量低于检测限。乳脂肪球蛋白酪蛋白的丰度如下：100R>100P>50P>sMEV-UC。通过测序分析在 sMEV-UC 中检测到超过 100 种成熟 microRNA，而在 EV-FF 中检测到 36 到 60 种 microRNA。只有 100R 和 sMEV-UC 产生了足够数量和质量用于测序分析的 mRNA；在 100R 和 sMEV-UC 中，平均有 276,000 和 838,000 个读数映射到大约 14,600 和 18,500 个基因。在主成分分析中，EV-FF 制剂中的 microRNA、mRNA 和蛋白质与对照 sMEV-UC 分开聚类。我们得出的结论是，在使用的条件下，流过滤产生了乳 EV 的异质群体。
