Comparative Analysis of Different Inactivation Methods on the Chemical and Bioactive Properties of Lactiplantibacillus Plantarum TAR4-Fermented Mulberry Leaf Extract

Lee, Wei En (2026) Comparative Analysis of Different Inactivation Methods on the Chemical and Bioactive Properties of Lactiplantibacillus Plantarum TAR4-Fermented Mulberry Leaf Extract. Final Year Project (Bachelor), Tunku Abdul Rahman University of Management and Technology.

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Abstract

Mulberry leaves (Morus alba) have gained popularity due to their rich bioactive constituents. While fermentation has been widely studied to enhance the functional potential of plant materials, the effect of post-fermentation inactivation on their bioactive properties remains underexplored. This study compared the effects of different inactivation methods on the physicochemical properties, chemical composition, and bioactivities of Lactiplantibacillus plantarum TAR4-fermented mulberry leaf extract (MLE). Mulberry leaves were fermented for 24 hours and subsequently subjected to thermosonication, thermal inactivation, and ultrasonication. Fermented extract without inactivation and unfermented extract served as the control and blank, respectively. The treated samples were analysed for pH, colour, titratable acidity (TA), total phenolic content (TPC), total flavonoid content (TFC), antioxidant activities (ABTS and FRAP), hypoglycaemic activity (α-amylase inhibitory), and advanced glycation end products inhibitory activity (AGEs). Additionally, LC-MS fingerprint was conducted to evaluate the trend of change in the phytochemical profile in MLE after fermentation and different microbial inactivation methods. Fermentation significantly lowered pH and raised TA, confirming acidification. Nevertheless, it increased TFC (from 11.08 ± 0.72 mg QE/mL to 6.87 ± 3.38 mg QE/mL) and AGEs significantly but decreased TPC from 80.43 ± 4.45 mg GAE/mL to 98.61 ± 3.81 mg GAE/mL. Despite thermal inactivation significantly increased TFC and FRAP, ABTS and α-amylase inhibition showed no significant changes across treatments (ABTS: range from 38.20 ± 4.28% to 43.20 ± 2.48%; α-amylase inhibition: range from 11.67 ± 1.93% to 17.80 ± 3.97%). The study proposes that ultrasonication best preserved AGEs (95.35 ± 0.01%), which is close to the control (96.00 ± 0.01%), while thermosonication caused the greatest loss of bioactivity. LC-MS fingerprint confirmed treatment dependent changes in the phytochemical profile, supporting the hypothesis that the inactivation method drives significant changes in chemical composition, resulting in altered bioactivity. This study provides insights into L. plantarum TAR4 fermentation enhances the bioactivity of MLE, and how the type of post fermentation inactivation method determines the extent to which these bioactivities are preserved. Future studies should optimize substrate preparation, refine inactivation parameters, and conduct metabolite profiling to identify and quantify compounds altered by each inactivation method.