Exopolysaccharide isolated from marine Bacillus subtilis strain AF2: production, characterization, purification and in vitro bioactivity

Abstract

Bacillus subtilis strain AF2 was obtained from the Mediterranean Sea and its identification was determined by its phylogenetic analysis of 16S rRNA sequences, as well as their morphological, physiological, and biochemical characteristics. The isolate yielded EPSS4 at a concentration of 7.4 g/l. EPSS4 constituted a significant proportion of the EPS recovered through purification using DEAE-Cellulose. The sample consisted of sulfate (24.71%) and uronic acid (18.55%). The fraction's viscosity was measured to be 1.1 mm²/sec, and the total hexose amine content was measured to be 9.06%. The monosaccharide composition of this fraction consists of glucose, fructose, xylose, and glucuronic acid in a molar ratio of 1.0:0.5:1.0:1.0, respectively. The toxicity studies on EPSS4 demonstrated its safety at 5 g/kg doses. EPSS4 underwent evaluation for its antioxidant, anticancer, and anti-inflammatory properties. The results indicated that the antioxidant activity percentages were as follows: 85.17 ± 0.77% for DPPH, 79.63 ± 1.05% for ABTS, 52.20 ± 0.67% for Fe²⁺ ion chelation ability, 75.80 ± 0.28% for Lipid peroxidation Inhibition capacity, 84.66 ± 1.21% for O^2- radicals scavenging capacity, and 55.09 ± 0.34% for NO scavenging capacity. The IC₅₀ value of EPSS4 for HepG-2 is 238.05 µg/ml. A-549 has determined the IC₅₀ values to be 102.71 µg/ml. The concentration of HCT-116 was measured to be 328.01 µg/ml, while the IC₅₀ value for the MCF-7 cell line was determined to be 204.77 µg/ml. The IC₅₀ values for the HEP-2 and PC-3 cell lines were 118.95 and 128.94 µg/ml, respectively. The anti-inflammatory activity of EPSS4 was assessed using various methods, including measuring its inhibitory effects on lipoxygenase (LOX) and cyclooxygenase (COX2). At a dosage of 100 µg/ml, EPSS4 had a LOX inhibitory activity of 80.54 ± 1.09% and a COX2 inhibitory activity of 84.70 ± 1.05%. Due to its antioxidant capabilities, the EPS produced from Bacillus subtilis strain AF2 is a promising option for application as a potent antioxidant agent in the healthcare system. So, from observed activities demonstrate that it has the potential to be employed as an anticancer, antioxidant and non-steroidal anti-inflammatory substance via improving adaptive immune responses.

Keywords: Bacillus subtilis; Anti-inflammatory; Antioxidant; Antitumor; Exopolysaccharide.