Antioxidant and probiotic potential of Lacticaseibacillus paracasei isolates from Malaysian water kefir grains

Water kefir is a traditional fermented beverage that comprises a microbially diverse community enriched in lactic acid bacteria (LAB). Previous studies have examined the predominant species recovered from each sample. However, functional strain-level variation among multiple LAB isolates within a single kefir grain source remains insufficiently explored. This study aimed to isolate and characterise Lactobacillus species from Malaysian water kefir grains and evaluate their antioxidant activity and probiotic potential. Bacterial isolates were initially characterized through colony morphology, Gram staining, and catalase testing. Antioxidant activity was assessed using 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging assays. Isolates demonstrating high levels of antioxidant activity were evaluated for probiotic characteristics, including acid and bile salt tolerance, adhesion to intestinal epithelial cells, and antibiotic susceptibility. Molecular identification was performed through 16S rRNA gene sequencing. Among fifteen isolates, isolates 2 and 5 exhibited significantly higher antioxidant activities (p < 0.05) with ABTS radical scavenging activities of 72.70 ± 3.38% and 77.74 ± 3.27%, and DPPH radical scavenging activities of 41.62 ± 1.18% and 48.07 ± 5.18%, respectively. Probiotic characterization demonstrated that isolate 2 exhibited higher bile tolerance and adhesion capacity with a 93.57 ± 1.31% survival rate under bile salt stress and 88.8 ± 6.3% adhesion rate to HT-29 cells. Molecular identification identified both isolates as Lacticaseibacillus paracasei, a species widely associated with probiotic functionality. Taken together, these findings highlight isolate 2 as a promising probiotic candidate with antioxidant potential, warranting further investigation to validate its functional efficacy and safety.