Date of Publication

2021

Document Type

Dissertation

Degree Name

Doctor of Philosophy in Biology

Subject Categories

Biology

College

College of Science

Department/Unit

Biology

Thesis Advisor

Esperanza C. Cabrera

Defense Panel Chair

Mariquit M. De Los Reyes

Defense Panel Member

Glenn G. Oyong
Llewelyn S. Moron
Renato G. Reyes
Sofronio P. Kalaw

Abstract/Summary

Lentinus species (Polyporaceae) are saprophytic, wood-rotting basidiomycetous mushrooms with significant nutritional and pharmacological values. They are important resources of functional food and bioactive metabolites. In this study, the molecular identity and variability, optimum mycelial submerged culture conditions and cytotoxicity of 15 Lentinus isolates collected from different areas in Luzon Island, Philippines were investigated. Genomic DNA from fresh Lentinus mycelia was extracted, PCR-amplified using ITS1F and ITS4BR primers, purified, sequenced. The sequence data were edited, aligned, and the consensus sequences were determined using BioEdit, and compared against NCBI GenBank database using BLASTn homology search. Phylogenetic trees were constructed using Maximum Likelihood method with Kimura-2-parameter model and Maximum Parsimony method in Molecular Evolutionary Genetics Analysis (MEGA X). The influence of liquid culture media, pH, temperature, illumination and agitation on the Lentinus mycelial biomass production was evaluated. Mycelial biomasses were mass produced, air-dried and extracted. Cytotoxicity of the ethanolic extracts of Lentinus mycelia against human colorectal carcinoma (HCT-116), hepatocellular carcinoma (HepG2), and normal kidney epithelial (HK-2) cell lines was determined using the CellTiter 96® Aqueous One Solution MTS proliferation assay. Results of BLASTn analysis of ITS1 and ITS4B consensus sequences confirmed the molecular identities of seven Lentinus species including Lentinus tigrinus, Lentinus squarrosulus, Lentinus sajor-caju, Lentinus strigosus, Lentinus swartzii, Lentinus glabratus and Panus (Lentinopanus) conchatus showing similarities that ranged from 97.90% to 100% (with 99% to 100% query coverage) with their corresponding GenBank sequences. Both maximum likelihood tree and most parsimonious tree supported the distinct grouping of isolates of each Lentinus species. Both phylogenetic trees consistently showed the separation of L. squarrosulus CPS5 from the other two L. squarrosulus isolates (LSQBot and LSQOs), and the separation of both L. strigosus isolates and P. conchatus from other Lentinus taxa, indicating their own separate lineage. Coconut water (CW) was the most suitable liquid culture medium for the maximum mycelial biomass production of all studied Lentinus. The optimum initial pH of CW for most of the isolates tested was found at pH 4.0 to 7.0. In terms of physical parameters, all Lentinus isolates preferred 30°C as the optimum temperature, indicating that they are tropical mushroom species. Lighted condition was found to be favorable for most Lentinus isolates. L. tigrinus BP32, L. squarrosulus LSQOs, L. sajor-caju LSCBot and C005, L. strigosus BIL1324, L. swartzii BIL4618, and P. conchatus A52 showed significantly higher biomass yield in static-flask culture condition while L. squarrosulus LSQBot exceedingly favored shake-flask culture condition. The mycelial biomass production of 15 Lentinus isolates was significantly increased by 1.2 to 1.7-fold when grown in the optimized submerged culture condition. Among Lentinus isolates, BIL4618 consistently recorded the highest biomass yield in all optimization parameters. Ethanolic extracts of Lentinus mycelia exhibited low cytotoxicity against HCT-116 human colorectal carcinoma cell line with IC50 values of 242.75 µg mL-1 to 444.79 µg mL-1. They were not cytotoxic to HepG2 hepatocellular carcinoma and HK-2 normal kidney epithelial cell lines with less than 50% cytotoxicity indices. In conclusion, the ITS region of rDNA is a useful genetic marker for molecular identification and species differentiation of Philippine Lentinus isolates. The mycelial biomass productivity of Lentinus may vary depending on the species and isolates, and this can be enhanced by growing them in their optimized submerged culture conditions, which is very essential in various nutraceutical, pharmaceutical and industrial applications. Furtherance of the cytotoxicity and other bioactivity profiling of Lentinus mycelial extracts is highly recommended.

Abstract Format

html

Language

English

Format

Electronic

Physical Description

xvii, 117 leaves

Keywords

Polyporaceae--Philippines; Lentinus; Species; Wild plants, Edible--Philippines; Mushrooms; Mycelium

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Embargo Period

9-21-2027

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Available for download on Tuesday, September 21, 2027

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