Optimization of fermentation media constituents for higher production of naringinase by <i>Paenibacillus stellifer</i> RAMCM-44 using Response Surface Methodology

Naringin is a flavonoid in citrus fruits and contributes to the bitterness of extracted citrus fruit juices. Naringinase has potential to hydrolyse the naringin into tasteless compounds and, therefore, reduces the bitterness of citrus juices. Majority of studies focused on naringinase production from fungal sources. The present study aimed to use Paenibacillus stellifer RAMCM-44 for the production of naringinase by optimization of media constituents using Response Surface Methodology (RSM) at the shake-flask level. Five media components were taken as variables with different concentrations to determine the optimal concentrations of media components. The five variables included peptone (0.5-1%, w/v), yeast extract (0.4-0.8%, w/v), NaCl (0.5-1%, w/v), naringin (0.1-0.25%, w/v) and KH2PO4 (0.5-1%, w/v), while MgSO4 (0.05%, w/v), MnSO4 (0.001%, w/v) and initial pH (6.5±0.2) during shake-flask fermentation, media were kept constant. The experimental data was analysed (RSM) and optimal levels of peptone (0.75%), yeast extract (0.6%), NaCl (0.75%), naringin (0.17%) and KH2PO4 (0.75%) were determined. The experimental values of naringinase activity (8.55 IU/ml) and biomass (0.826 OD600) closely matched the predicted values. The criteria, including analysis of variance (ANOVA), R values, coefficient of variance and model significance were assessed. The R-value for naringinase production was found to be 0.9976, while the model F-value was 228.74 and the lack of fit F-value was 109.77 for naringinase activity, revealing the current study's significance. This is the first report on optimising media components and applying RSM for the production of naringinase by P. stellifer RAMCM-44, which could benefit studies involving scale-up.