Effects of Bacillus subtilis var. natto Fermentation and the Addition of Mulberry Leaf Extract and Mulberry Juice on the Chemical and Bioactive Properties of Okara

Khong, Yuet Kei (2025) Effects of Bacillus subtilis var. natto Fermentation and the Addition of Mulberry Leaf Extract and Mulberry Juice on the Chemical and Bioactive Properties of Okara. Final Year Project (Bachelor), Tunku Abdul Rahman University of Management and Technology.

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Abstract

Okara, a protein-rich byproduct of soymilk production, and mulberry leaves and fruit, rich in various bioactive substances, have gained interest for their potential health benefits. This study investigated the effects of B. subtilis var. natto fermentation and the addition of mulberry leaf extract (MLE) and mulberry juice (MJ) on the chemical and bioactive properties of okara. MLE was prepared via cellulase-assisted ultrasound extraction, and MJ was extracted from rehydrated dried fruit. Okara (50 %) in distilled water (O+DW) was used as experiment reference, while samples with 50 % of MLE or MJ in 50 % of okara (O+MLE/O+MJ) were prepared. All samples were fermented with B. subtilis at 37 °C for 24 hours. Unfermented samples served as controls. pH, total crude protein content of solid residues, total soluble protein, total phenolic content (TPC), total flavonoid content (TFC), antioxidant activity (ABTS, FRAP), anti-lipoxygenase activity (anti-LOX), α-amylase inhibition activity, and anti-thrombolytic activity were measured post-fermentation. Significant changes (p<0.05) in pH and TPC were observed in all samples. The anti-thrombolytic activity only significant in O+MLE and O+MJ samples after fermentation, while the crude protein content changes were significant only in O+DW. TFC and α-amylase inhibition activity decreased in O+MJ, while anti-LOX decreased in O+MLE and increased in O+DW. These findings suggest that both MLE and MJ significantly influenced B. subtilis fermentation due to their phytochemical content. The addition of MJ and MLE led to increased TPC, TFC, FRAP, and anti-LOX in okara. Control O+MJ exhibited the highest antioxidant and anti-inflammatory activities, while B. subtilis fermentation negatively impacted the bioactivity of MLE and MJ samples. However, B. subtilis fermentation significantly increased TPC, TFC and anti-LOX in O+DW. Future research should explore sequential fermentation with MJ addition to optimize the bioactivity and nutrient profile of okara.