Chitosan based ecofriendly nanoencapsulation formulation of Clitoria ternatea extract coated bacterial probiotic strain Lactobacillus acidophilus: A Sustainable Approach to Improve Probiotic Functionality, Stability, and Tolerance

Abstract

In this study, a sustainable nanoencapsulation formulation of Clitoria ternatea extract-coated bacterial probiotic strain Lactobacillus acidophilus using nanoscale chitosan (CT-CS-LA-NC) was developed to enhance probiotic functionality, stability, and tolerance. The structural and functional characteristics of the nanoencapsules were analyzed using Scanning Electron Microscopy (SEM), Fourier Transform Infrared Spectroscopy (FTIR), and Thermogravimetric Analysis (TGA). These analyses confirmed that the CT-CS-LA-NC nanoencapsules were nanoscale in size, highly thermostable, and exhibited strong interactions among the polymer matrix, bacterial cell surface components, and plant extract. The encapsulation efficiency was found to be 98.43%, indicating effective preservation of viable probiotic cells during the encapsulation process.The antibacterial activity of CT-CS-LA-NC demonstrated significant inhibition against Escherichia coli and Bacillus subtilis. Antioxidant assessment revealed a free radical scavenging activity of 36.46% at 80 µg/mL. Under simulated gastric conditions (pH 2.5 and 3.5), the survivability of free and nanoencapsulated L. acidophilus showed notable differences after 36 hours. The encapsulated cells exhibited a higher survival rate of 60.93 ± 0.68% at pH 2.5, compared to only 21.29 ± 0.13% for free cells. Similarly, under bile tolerance conditions, encapsulated cells showed a survivability of 39.46 ± 0.68% at pH 2.5.Storage stability studies at 4°C over 28 days revealed a rapid decline in the viability of free L. acidophilus cells, whereas CT-CS-LA-NC encapsulated cells maintained 94% viability after 7 days and 52% after 28 days. Molecular docking analysis using CB-Dock demonstrated strong binding affinities between anthocyanin, lactic acid, and chitosan ligands with gut-associated proteins such as O-GlcNAcase (7K41) and S-layer associated protein (SlpA) (8AE1), showing binding energies of –9.4 and –9.6 kcal/mol, respectively. Overall, the biocompatible and environmentally safe CT-CS-LA-NC formulation significantly improved probiotic stability during storage, enhanced survivability under harsh gastrointestinal conditions, and prolonged probiotic shelf life. These findings highlight its potential application as a sustainable ingredient for functional foods and nutraceutical formulations.