Media formulation using statistical methodology to enhance α-amylase production for green synthesis of Au-NPs by Bacillus subtilis VSP4 under solid-state fermentation

Vimalkumar S Prajapati; Vaibhavkumar N Mehta; Swati K Patel

doi:10.3389/fbioe.2025.1569902

Media formulation using statistical methodology to enhance α-amylase production for green synthesis of Au-NPs by Bacillus subtilis VSP4 under solid-state fermentation

Front Bioeng Biotechnol. 2025 Jun 3:13:1569902. doi: 10.3389/fbioe.2025.1569902. eCollection 2025.

Authors

Vimalkumar S Prajapati¹, Vaibhavkumar N Mehta², Swati K Patel³

Affiliations

¹ Division of Microbial and Environmental Biotechnology, ASPEE SHAKILAM Biotechnology Institute (ASBI), Navsari Agricultural University, Surat, Gujarat, India.
² Center of Excellent in Nano Agri-Biotechnology, ASPEE SHAKILAM Biotechnology Institute (ASBI), Navsari Agricultural University, Surat, Gujarat, India.
³ Division of Plant Biotechnology, ASPEE SHAKILAM Biotechnology Institute (ASBI), Navsari Agricultural University, Surat, Gujarat, India.

Abstract

In recent years, gold nanoparticles (Au-NPs) have garnered popularity for their remarkable and promising applications in various areas. Here, we report the synthesis of Au-NPs using extracellular amylase produced by Bacillus subtilis VSP4 under solid-state fermentation (SSF) through the reduction of AuCl₄ with retention of enzymatic activity in the complex. Accordingly, B. subtilis VSP4 was exploited to enhance α-amylase production under SSF using the Plackett-Burman design, followed by the central composite design (CCD) of response surface methodology (RSM). According to our analysis, the most significant components in the medium are starch, yeast extract, and CaCl₂ (significance >95%, ANOVA), which prominently enhance enzyme production. The optimum levels of these three selected variables were evaluated using CCD-RSM (20 runs), and it was confirmed that 0.05 g of starch, 0.1 g of yeast extract, and 5 mM of CaCl₂ per 5 g of wheat bran under SSF produced the maximum α-amylase yield (169.72 U/gds). The F-value of the quadratic model (18.36) implies that the model is significant, while the F-value of the lack of fit (3.17) indicates that the lack of fit is not significant, meaning that the model has good fit. The coefficient of variance was found to be 0.369, which denotes that the experiments performed herein are reliable (R² = 0.94) (multiple correlation coefficient), and the standard deviation for the quadratic model was found to be 4.72. We also performed separate validation experiments to confirm the adequacy of the quadratic model. The present work highlights α-amylase production by B. subtilis VSP4 under SSF, which was prominently enhanced by adopting a statistical experimental design, leading to the formation of Au-NPs of average size 5.17 ± 0.85 nm showing a surface plasmon resonance peak at 528 nm.

Keywords: Plackett–Burman design; central composite design; gold nanoparticles; response surface methodology; solid-state fermentation; α-amylase production.