Due to the live nature of probiotics, changes in the manufacturing processes or facilities can result in differences in the product itself (Sanders et al, Ann N Y Acad Sci. 2014;1309:1–18). Recently, under the brand VSL#3, a formulation produced by a manufacturer different from the previous one, has been commercialized in some European Countries. We compared the VSL#3 produced in USA with VSL#3 produced in Italy and found that the biological effects of the 2 products when compared in vitro on tumor cell lines' viability, proliferation, cell cycle profile and apoptotic death levels were significantly different (Cinque et al, PLoS One. 2016, In press). Here we report additional data regarding the physical, chemical and biological characteristics of the 2 formulations available in Europe.Methods:
Samples of VSL#3 were biophysico-chemically characterized from their solid state by differential scanning calorimetry and thermogravimetry analysis and for their kinetics of stability, cell size (hydrodynamic diameter) and zeta potential (electrophoretic mobility) in aqueous dispersion. The live/dead status of bacteria in the VSL#3 product was assessed using a mixture of SYTO®13 green fluorescent nucleic acid stain and the red fluorescent nucleic acid stain, propidium iodide. Cells were inspected visually using an epifluorescence microscope. Stocks of 1 g (102 billion bacteria) of each VSL#3 lot were suspended in 10 mL culture medium and incubated in a thermomixer, with shaking at 37°C for 2 hours. Intestinal epithelial (IEC-6) cells were plated onto polylysine coated coverslips. Thirty thousand cells were incubated in the presence or absence of bacterial suspension (1000 live bacterial cells/cell) for 24 hours. Afterward the cells were observed using a light microscopy or prepared for scanning electron microscope observation.Results:
Both powder samples showed similar melting characteristics with Tm around 116°C but slight enthalpy difference. Their decomposition/degradation were different; the USA product decomposed/degraded faster than the Italy product with temperature increase in the range of 170 to 310°C. In aqueous dispersion, the kinetics of aggregation/sedimentation as well as cell hydrodynamic diameters appeared different. The USA product exhibited a larger hydrodynamic average size and aggregated/decanted faster than the Italy product. Cell biological behavior information showed a low number (16%) of viable bacterial cells in the Italy product compared to the USA product (76%), both products at 8 months shelf life. Morphological differences between IEC-6 cell cultures treated with both VSL#3 samples were also evidenced through optical and scanning electron microscopy.Conclusions:
In conclusion, the VSL#3 made in Italy is quite different from the VSL#3 made in USA. Different biophysico-chemical characteristics and reduced number of viable bacterial cells impact on the biological profile of the product.