Date of Graduation

5-2022

Document Type

Dissertation

Degree Name

Doctor of Philosophy in Cell & Molecular Biology (PhD)

Degree Level

Graduate

Department

Cell & Molecular Biology

Advisor/Mentor

Hettiarachchy, Navam S.

Committee Member

Thallapuranam, Suresh

Second Committee Member

Kral, Timothy A.

Third Committee Member

Lay, Jackson O. Jr.

Fourth Committee Member

Carbonero, Franck G.

Keywords

Anti-melanogenesis; Bacillus subtilis (natto); Bioactive peptide; Protein extraction; Rice bran; Solid state fermentation

Abstract

Heat-stabilized defatted rice bran (HDRB) is an abundant and inexpensive agro-industrial by-product of rice milling and rice oil extraction process. Furthermore, HDRB is a potential source of proteins. However, the direct protein extraction has low yield of protein due to the impact of heat stabilization and oil extraction process which made the HDRB proteins bound and interact with other components. This research is based on using solid-state fermentation (SSF), which is a natural and cost-effective approach to facilitate protein extraction from HDRB using Bacillus subtilis (natto) Takahashi as a microorganism.

Response surface methodology (RSM) analysis was used with Box-Behnken design (BBD) to optimize SSF conditions for extraction of the maximum water-soluble proteins and peptides (WSPP) from HDRB, and the predicted optimum conditions were 40.96% w/v water content, 6.27 Log CFU/g HDRB inoculum log, and 61.01h fermentation time. Conducting SSF under the optimized conditions facilitated extraction of 64.6 ±0.7% of total protein in HDRB as WSPP in comparison to 20.6% of protein extracted from nonfermented HDRB. Besides, the SSF increased the amount of low molecular weight of protein fractions and peptides with size of <5KDa.

The WSPP of SSF-HDRB extract was fractionated into molecular size ranges of >50, 10-50, 5-10, and(UF) technique, and the fractions were evaluated for tyrosinase, the main enzyme in melanogenesis pathway, and elastase inhibition activities. The results showed that the highest in-vitro elastase and tyrosinase inhibition activities by UF peptide fraction < 5Kda and were 12.9% and 68.5%, respectively. Further separation of fraction

The SEC fractions (F4 and F5) were characterized and purified by reverse phase HPLC using C-18 column and among the collected fractions three fractions, namely F8, F14, and F40, showed enhanced anti-tyrosinase activity of IC50 values of (870.3, 445.8, and 187.6 µg/mL) respectively, with dose dependent inhibition. Also, the fractions showed potential inhibition of melanogenesis activity without cytotoxic effect in mouse B16F10 melanoma cells, and peptide fraction (F40) had the greatest melanogenesis inhibition activity (84.2% ±1.3) and IC50 value (235.2 µg/mL) signifying the potential of F40 as a cosmeceutical agent for treatment of melanin-related skin disorder.

The most abundant peaks in the three fractions were identified by mass spectrometry and proteomic tools using rice proteomic database. Five peptides were identified having the amino acid sequence (TSFTL) and (APDLPML) from (F40), (FPLHF) and (SYVFGCF) from (F14), and (SCADGGF) from (F8). WSPP of SSF-HDRB has potential antioxidant activities, and showed (56.0, 44.4, and 84.5 %) of DPPH free radical, superoxide radicals, and hydroxyl radicals scavenging activities, respectively.

Based on the results, bacterial SSF using the probiotic B. subtilis (natto) Takahashi is an efficient approach to enhance extraction of WSPP from HDRB with potential bioactivities and add-value to HDRB in finding applications as a functional ingredient in the cosmeceutical products that support skin health and control the oxidative stability in food products.

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