Neuroprotective potential of optimized <i>Lactobacillus fermentum</i> extracts against Acetylcholinesterase activity in Zebrafish model and cytotoxicity studies in PC12 cell line

Alzheimer’s disease is linked with reduced levels of acetylcholine due to elevated acetylcholinesterase (AChE) activity. Antioxidants and natural AChE inhibitors (AChEIs) are becoming more popular due to their potential neuroprotective benefits. The present work aimed to enhance the production of AChE inhibitors by Lactobacillus fermentum using phytogenic substrates and evaluate their neuroprotective effects in a zebrafish model. Media optimization was carried out using response surface methodology (RSM) with Cinnamomum cassia (0.5 mg/mL), Syzygium aromaticum (1 mg/mL), and Phoenix dactylifera seed (1 mg/mL) as substrates. AChE inhibition was predicted using a quadratic model (R2 = 97.61%), and the maximal inhibition of 28.69% closely matched the expected value of 28.56% (Run no 11). The optimized L. fermentum chloroform extract (OLFCE) exhibited a strong antioxidant potential, with 75.09% DPPH scavenging activity. OLFCE exhibited minimal cytotoxicity on H2O2-induced PC12 cell line, maintaining 95–99% cell viability across all tested concentrations. Treatment of zebrafish with 2.5–10 mg/mL OLFCE significantly enhanced antioxidant enzyme activities (superoxide dismutase and catalase) and decreased AChE levels in brain, liver, and plasma. GC-MS analysis of OLFCE revealed high levels of potential AChEIs i.e. 3,4-dimethyl benzaldehyde (66.81%) and phenol, 2,4-bis(1,1-dimethylethyl) (16.84%). This study demonstrates, for the first time, that substrate-optimized L. fermentum can produce potent AChEIs and antioxidant compounds, as validated through in vivo zebrafish assays. The findings suggest that OLFCE has promising Acetylcholinesterase Inhibitor activity, which can be used for the treatment of Alzheimer’s Disease in the future.