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Isolation of low molecular mass antibacterial compound from supermeal worm Zophobas Morio fabricius
International Islamic University Malaysia
Kulliyyah of Engineering, International Islamic University Malaysia
Lower molecular mass (< 1 kDa) insect antimicrobial compounds play a major role as the defensive armament against deadly microbial invasions. Their potent inhibitory effects display high potentials in many therapeutic applications and developments. The aim of this research is to explore the extraction, optimization of the process extraction conditions and partial purification of such compound from Zophobas morio supermealworms. Larvae were subjected to a solvent screening study incorporating acidified hydro-alcoholic extraction using water, methanol, ethanol, isopropanol and butanol to select the best solvent system based on the formation of large inhibition zone on bacteria in the susceptibility assay. Factors affecting the process extraction conditions including homogenizing time; solid-to-solvent ratio and temperature were statistically optimized using Response Surface Methodology (RSM) for maximal recovery. The antibacterial compound, collectively known as Zm-NI factor was partially purified by the bioassay-guided solid phase extraction on the reversed-phase and normal phase columns and was further analyzed by the analytical profiling. Isopropanol (90:9:1,v/v/v) was selected as the best solvent for Zm-NI factor recovery as its extract produced significantly (p < 0.05) larger inhibition zones on S. aureus (IMR: S822) (15.70 ± 0.33 mm), E. coli (IMR: E255) (14.53 ± 1.53 mm), K. pneumoniae (IMR: K36) (14.00 ± 1.00 mm) and P. aeruginosa (IMR: P167) (13.30 ± 0.58 mm) compared to other solvent extracts. S. aureus (IMR: S822) was the most susceptible towards the isopropanolic extract. Zm-NI factor exhibited a broad-spectrum antibacterial activity. It is a constitutive, non-viable, polar molecule with high resistance to trypsin, actively stable at high temperature and wide pH range. Its antibacterial activity is negatively affected by EDTA, SDS and β-mercaptoethanol. The optimization of the extraction process conditions by a Box-Behnken design (BBD) at a fixed isopropanol concentration revealed that the model could not generate an optimized extraction condition due to the occurrence of negative interactions between the test variables. At any high level of homogenizing time and temperature, a maximal recovery of Zm-NI factor could be achieved with the use of high ratios of isopropanol. Partial purification of Zm-NI factor resulted in the fractionation of the compound in 25 samples with strong antibacterial activity by normal phase mode. Analytical profiling of the semi-purified sample by UHPLC-MS indicated a total of 49 distinct molecules with molecular mass less than 1 kDa, deemed to be the target candidates (active fractions) of the compound. The isolation of Zm-NI factor from supermealworms implies the possibility of its development as a novel antibiotic to combat antibiotic resistance. Further studies should involve the analytical purifications and characterizations of the compound. The determination of an optimum Zm-NI factor extraction condition is also essential to provide a basis for scaling-up as to be used in the pilot scale production in the future.
Thesis (MSBTE)--International Islamic University Malaysia, 2015.
Includes bibliographical references (leaves 185-224).