Isolation, Phenotypic Screening, and Extracellular Serotonin-Like Immunoreactivity of Presumptive Lactic Acid Bacteria from Butterfly Pea Flower (Clitoria ternatea L.) Kombucha

Abstract

Background: Major depressive disorder is a leading cause of disability, and interest in the microbiota-gut-brain axis has stimulated bioprospecting of food-derived lactic acid bacteria (LAB) for neuroactive metabolite production.

Objective: To isolate and phenotypically screen acidogenic bacteria from butterfly pea flower (Clitoria ternatea L.) kombucha and to evaluate their cell-free supernatants for extracellular serotonin-like immunoreactivity.

Methods: Colonies were isolated from day-14 kombucha on MRS agar supplemented with 1.0% CaCO₃ and characterised by Gram staining, catalase testing and broth acidification in MRS broth (24 h, 37 °C). Serotonin-like immunoreactivity in cell-free supernatants was quantified by a competitive enzyme-linked immunosorbent assay (ELISA) calibrated with a four-parameter logistic (4-PL) model.

Results: Three isolates (Isolates 3, 6 and 8) were presumptive LAB (Gram-positive, catalase-negative bacilli), dissolved CaCO₃ (clear zones 14.8–18.4 mm) and reduced broth pH from 7.00 to 3.94–4.15. The 4-PL model returned R² = 0.9993 and back-calculated the standards to within 0.1% of nominal. Cell-free supernatants gave serotonin-like immunoreactivity averaging 18.11 ± 4.54 ng/mL (Isolate 3 highest, 23.13 ng/mL) versus 1.28 ± 0.67 ng/mL in sterile media (exploratory Welch's t = 6.34, df = 2.09, p = 0.022).

Conclusion: Presumptive lactic acid bacteria from butterfly pea flower kombucha yielded a measurable serotonin-like immunoreactivity signal in culture broth. These preliminary in vitro findings warrant 16S rRNA gene sequencing, chromatographic confirmation of analyte identity (HPLC/LC-MS) and in vivo evaluation.