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The objectives of this study were to determine the optimal extraction condition of cassava pulp and their effects on mercury bioaccessibility and bioavailability. The extraction process requires the starch be separated from the fiber by enzyme application. The enzyme reaction conditions for the solubilization were optimized via a response surface methodology (RSM). The selected dependent variable was percentage of neutral detergent fiber (NDF). The highest NDF (79.68%) of crude dietary fiber could be gotten from enzymatic digestion condition of 0.1% of α-amylase (w/v), 0.1% of amyloglucosidase (v/v) and 1% of neutrase (v/v). In addition, NDF affecting the mercury bioavailability was estimated by using in vitro digestion and Caco-2 human intestinal cell model system. In vitro digestion (bioaccessibility) showed that the fiber could reduce mercury bioaccessibility to 2-57% compared with the control (0-1000 mg of NDF in 1 g of fish tissue) in a dose dependent manner. The effect of fish tissue amount (0 - 4 g) on mercury quantification when 500 mg of NDF was added in digestion model test showed that the NDF did assist with reduction of mercury amount in fish tissue from 39% to 21% compared with control (the control lacks NDF). Furthermore, the Caco-2 cell was utilized for evaluation of intestinal cell accumulation and supporting reliable estimating bioavailability. The results showed that the mercury transfer to intracellular range from 9.07-5.97% for control and 6.54- 4.63% in the media containing 500 mg NDF. In conclusion, this study suggests that NDF prepared from cassava pulp could decrease mercury bioavailability by inhibiting the mercury transfer to the aqueous fraction and could be applied in functional food and dietary supplement products. [ABSTRACT FROM AUTHOR]