title: Microbial community and health benefits of Traditional soaked Rice (Panta Bhat) in Bangladesh
design: Microbial community and health benefits of Traditional soaked Rice (Panta Bhat) in Bangladesh
library_construction_protocol: Soaked Rice (Panta Bhat) was prepared following traditional Bangladeshi methods. In brief, whole Basmati rice grains, collected from a local supermarket, were washed and cooked with water at 100 °C for 20-30 minutes in an aluminium pan and then cooled to room temperature. Rice was separated in two clean sterile mud pots, maintaining sterile conditions by mixing rice with water using sterile utensils. Then all the rice soaked in water was kept overnight at room temperature for 12 to 14 hours for further analysis. The rice was prepared by the individual participants for their own consumption as per the ethical approval. DNA from rice samples soaked at 20 °C and 30 °C was extracted using the DNeasy® PowerFood® Microbial Kit, following a modified protocol designed for optimal DNA yield as per the manufacturer's guidelines. Initially, 0.25 g of soaked rice sample was combined with 1.8 ml of rice broth in a 2 ml microcentrifuge tube. The samples were homogenized using a FastPrep-24™ 5G Homogenizer at a speed of 8 m/s for 6 mins.  The resulting microbial food culture was centrifuged, and the supernatant was removed. This process was repeated until 10 ml of soaked rice broth was centrifuged to obtain a concentrated rice pellet.  The cell pellet was resuspended in 450 µl of pre-warmed (55°C) MBL solution and transferred to a PowerBead Tube. Instead of using a vortex adapter, the PowerBead Tubes were placed in the FastPrep-24™ 5G Homogenizer, vortexed horizontally for 10 min at maximum speed. After centrifugation, the supernatant was transferred to a clean tube, and IRS solution was added and incubated before another centrifugation step. The resulting supernatant was mixed with MR solution and loaded onto an MB Spin Column. After several wash steps, the purified DNA was eluted using 50 µl of elution buffer (EB).  The purified DNA samples were quantified using a NanoDropTM ( One Microvolume UV-Vis Spectrophotometer, ThermoScientific). Samples with a concentration of at least 10 ng/µl were selected and sent to Novogene (Genomic services, Cambridge, UK) for sequencing. The V4 region of the 16S rRNA gene was amplified using primers 515F (5' GTGCCAGCMGCCGCGGTAA 3') and 806R (5' GGACTACHVGGGTWTCTAAT 3').