所有王国的细胞都会产生细胞外囊泡(EV);因此,它们存在于大多数环境和体液中。副干酪乳杆菌产生具有附着的生物活性蛋白(P40和P75)的EV。在这项研究中,在五种携带副干酪乳杆菌的不同商业乳制品发酵产品中发现了EV和功能蛋白。分离出这些产品中存在的菌株,除了一个例外,所有生产的小型电动汽车(24-47d.nm)携带P40和P75。为了从牛奶EV中消灭细菌EV,产品在15,000×g(15K)下进行离心分馏,33,000×g(33K),和100,000×g(100K)。P75存在于所有上清液和颗粒中,但是P40仅在与15和33K颗粒结合的两种产品中发现,可以从所有100K电动汽车中扩增出副干酪乳杆菌的16SrDNA,表明存在副干酪EV。为了研究细菌EV和蛋白质与牛奶EV的相互作用,将副干酪乳杆菌BL23EV添加到三种商业UHT奶制品中。在100K离心的样品中发现了类似于副干酪BL23EV的小尺寸囊泡(50-60d.nm),但有趣的是,P40和P75在15和33K颗粒中与EV结合,含有较大尺寸的牛乳EV(200-300d.nm)。对从EV扩增的16SrDNA条带进行测序证明了牛奶和发酵产品中存在不同来源的细菌EV。此外,副干酪乳杆菌16SrDNA可以用所有样品的物种特异性引物扩增,显示所有EV部分(15、33和100K)中存在副干酪乳杆菌EV,这表明这些细菌EV可能会聚集并与牛奶中的EV共同分离。P40和P75蛋白将与特定人群的牛奶EV(15和33K)相互作用,因为它们在发酵产品和牛奶中被检测到与它们结合,这可能迫使部分副干酪乳杆菌在较低的离心力下沉降。这项研究解决了技术上复杂的问题和基本问题,这将促进新的研究,重点关注发酵过程中益生菌的分子行为以及介导发酵产品健康益处的作用机制。
Cells of all kingdoms produce extracellular vesicles (EVs); hence, they are present in most environments and body fluids. Lacticaseibacillus paracasei produces EVs that have attached biologically active proteins (
P40 and P75). In this study, EV and functional proteins were found in five different commercial dairy-fermented products carrying L. paracasei. Strains present in those products were isolated, and with one exception, all produced small EVs (24-47 d.nm) carrying
P40 and P75. In order to winnow bacterial EV from milk EV, products were subjected to centrifugal fractionation at 15,000 × g (15 K), 33,000 × g (33 K), and 100,000 × g (100 K). P75 was present in all supernatants and pellets, but
P40 was only found in two products bound to the 15 and 33 K pellets, and 16S rDNA of L. paracasei could be amplified from all 100 K EVs, indicating the presence of L. paracasei EV. To investigate the interactions of bacterial EV and proteins with milk EV, L. paracasei BL23 EV was added to three commercial UHT milk products. Small-size vesicles (50-60 d.nm) similar to L. paracasei BL23 EV were found in samples from 100 K centrifugations, but intriguingly,
P40 and P75 were bound to EV in 15 and 33 K pellets, containing bovine milk EV of larger size (200-300 d.nm). Sequencing 16S rDNA bands amplified from EV evidenced the presence of bacterial EVs of diverse origins in milk and fermented products. Furthermore, L. paracasei 16S rDNA could be amplified with species-specific primers from all samples, showing the presence of L. paracasei EV in all EV fractions (15, 33, and 100 K), suggesting that these bacterial EVs possibly aggregate and are co-isolated with EV from milk.
P40 and P75 proteins would be interacting with specific populations of milk EV (15 and 33 K) because they were detected bound to them in fermented products and milk, and this possibly forced the sedimentation of part of L. paracasei EV at lower centrifugal forces. This study has solved technically complex problems and essential questions which will facilitate new research focusing on the molecular behavior of probiotics during fermentation and the mechanisms of action mediating the health benefits of fermented products.