# Valorization of Riceberry Broken Rice and Soybean Meal for Optimized Production of Multifunctional Exopolysaccharide by Bacillus tequilensis PS21 with Potent Bioactivities Using Response Surface Methodology

## TL;DR

This study uses rice and soybean by-products to produce a valuable exopolysaccharide with antioxidant, antimicrobial, and anticancer properties.

## Contribution

A novel method for high-yield EPS production using agro-industrial by-products and optimized conditions for bioactivity enhancement.

## Key findings

- Bacillus tequilensis PS21 produced 39.82 g/L EPS using optimized RBR and SBM substrates.
- The EPS showed strong antioxidant, antimicrobial, and anticancer activities with IC50 values of ~225 µg/mL.
- The EPS is a homopolysaccharide with glucose monomers and thermal stability up to ~250 °C.

## Abstract

This study explores the valorization of agro-industrial by-products—riceberry broken rice (RBR) and soybean meal (SBM)—as cost-effective substrates for enhancing exopolysaccharide (EPS) production by Bacillus tequilensis PS21. Eight Bacillus strains were screened, and B. tequilensis PS21 demonstrated the highest EPS yield (2.54 g/100 mL DW). The EPS displayed a strong antioxidant capacity with 65.5% DPPH and 80.5% hydroxyl radical scavenging, and a FRAP value of 6.51 mg Fe2+/g DW. Antimicrobial testing showed inhibition zones up to 10.07 mm against Streptococcus agalactiae and 7.83 mm against Staphylococcus aureus. Optimization using central composite design (CCD) and the response surface methodology (RSM) revealed the best production at 5% (w/v) RBR, 3% (w/v) SBM, pH 6.66, and 39.51 °C, yielding 39.82 g/L EPS. This EPS is a moderate-molecular-weight (11,282 Da) homopolysaccharide with glucose monomers. X-ray diffraction (XRD) showed an amorphous pattern, favorable for solubility in biological applications. Thermogravimetric analysis (TGA) demonstrated thermal stability up to ~250 °C, supporting its suitability for high-temperature processing. EPS also exhibited anticancer activity with IC50 values of 226.60 µg/mL (MCF-7) and 224.30 µg/mL (HeLa) at 72 h, reduced colony formation, inhibited cell migration, and demonstrated anti-tyrosinase, anti-collagenase, and anti-elastase effects. This study demonstrates the successful valorization of agro-industrial by-products—RBR and SBM—for the high-yield production of multifunctional EPS with potent antioxidant, antimicrobial, and anticancer properties. The findings highlight the sustainable potential of these low-cost substrates in supporting the development of green and value-added bioproducts, with promising utilizations across the food, pharmaceutical, and cosmetic sectors.