American Federation for Medical Research

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Extraction of Extracellular Vesicles from a Fermented Beverage: A Potential Gut-brain Link
Apoorva Vashisht, Steve Mathew, Allison B. Reiss, James H. Grendell, Lora J. Kasselman. Biomedical Research, NYU Winthrop Hospital, Mineola, New York, United States

Purpose of Study Extracellular Vesicles (EVs), like microvesicles and exosomes, have the ability to transfer cargo such as RNA to target cells such as neurons and microglia because they can cross the blood brain barrier (BBB). Thus, EVs provide a potential therapeutic approach to neurological disorders. mRNA, microRNA, lipids and proteins can be transported between cells via exosome-mediated mechanisms. EVs are shed from both prokaryotic and eukaryotic cells, including probiotic organisms. Probiotics have been linked to improvement in mental health, but the mechanisms are unclear. The purpose of this study is to determine whether EVs can be extracted from a fermented beverage containing probiotic organisms.
Methods Used Water kefir, a non-dairy fermented beverage, was cultured using kefir grains, sugar, molasses and diH2O. Culture supernatant was collected and super-centrifuged at 11,000 x g for 13 min. Supernatant was then collected and spun at 13,000 x g for 15 min. The supernatant was then vacuum-filtered through a 0.1um membrane. The filtrate was filtered through 0.45um membrane using syringe filters. The filtrate was super-centrifuged at 38,400 x g for 2 hours. The pellet was resuspended in sterile PBS and then ultra-centrifuged at 100,000 x g for 1 hour. The pellet was resuspended in 100μl of sterile PBS to obtain the EV preparation. The EVs were then stained with Alexa Fluor 488 membrane-dye and visualized using a fluorescent Nikon microscope.
Summary of Results We successfuly extracted EVs from the non-dairy fermented beverage water kefir and were able to visualize them using fluorescent microscopy. This is the first report of successful isolation of EVs from kefir, a food containing a complex microbial community that is commonly consumed in the human diet.
Conclusions Isolation of EVs from a fermented beverage will allow the study of the interaction between microbiota in the digestive system and the brain. The delivery of probiotic EV-derived cargo from the gut to the central nervous system across the BBB may be one mechanisms explaining the positive impact of probiotics on mental health. Analysis of EV content and cell culture studies of kefir-derived EVs and human neurons and microglia are ongoing and can elucidate the gene expression and signaling pathways in these cells that are impacted by EVs.


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