Evaluation of efficiency of Methods used for extraction of Bifidobacterium bifidum-Derived Extracellular Vesicles: An experimental study

Bacteria naturally secret nano-scale vesicles containing a wide range of biomolecules, such as proteins, DNA,
and RNA. These vesicles are called extracellular vesicles (EVs). EVs play important roles in host-microbiota interactions. For isolating
EVs, different methods have been proposed and each method has its advantages and also limitations. Therefore, in the current study,
efficacy of two methods used for extraction of EVs was investigated.
Materials & Methods: For this purpose, Bifidobacterium bifidum was cultured in MRS broth under anaerobic conditions. In the first
isolation protocol, ultra-centrifugation was used (Ultra-method) and in the second protocol, ultra-centrifugation (Non-Ultra method)
was not used. After isolation, protein content was measured by the NanoDrop system. Sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) technique was utilized to compare protein pattern of the EVs. Scanning electron microscopy (SEM)
images of the EVs҆ samples were taken and size of the EVs was evaluated by Digimizer software.
Results: The results showed that the EVs isolated by the Ultra-method had significantly higher vesicle-associated protein content
compared to those isolated by the Non-Ultra method (3.42 and 0.26 mg/ml, respectively). More and larger EVs (up to 235 nm and with
frequent size ranging between 100 – 125 nm) were isolated by the Ultra-method compared to the Non-Ultra method (up to 117 nm and
with frequent size ranging between 50–75 nm). Also, protein patterns of the EVs were similar in both methods and protein bands were
observed at 25 to 250 KDa in both methods.
Conclusion: Our results showed that ultra-centrifugation method is a more proper method for isolation of B. bifidum-derived EVs and
produces a higher amount of EVs with higher protein content and proper sizes. However, further studies are required to confirm our
results.