Isolation, purification and characterization of anti-fungal peptides from super meal worm (Zophobas morio Fabricus) /
In the last few years, fungal infection has increased outstandingly and this has been blamed for the rise in the number of deaths in patients with compromised immune systems. Commercially, antifungal remedy from natural sources is limited. In this study, antifungal peptides have been recovered from...
Saved in:
| Main Author: | |
|---|---|
| Format: | Thesis |
| Language: | English |
| Published: |
Kuala Lumpur :
Kulliyyah of Engineering, International Islamic University Malaysia,
2017
|
| Subjects: | |
| Online Access: | Click here to view 1st 24 pages of the thesis. Members can view fulltext at the specified PCs in the library. |
| Tags: |
Add Tag
No Tags, Be the first to tag this record!
|
| Summary: | In the last few years, fungal infection has increased outstandingly and this has been blamed for the rise in the number of deaths in patients with compromised immune systems. Commercially, antifungal remedy from natural sources is limited. In this study, antifungal peptides have been recovered from whole body extraction of fourth instar larvae of Zophobas morio Fabricus. The aim of this study is to screen the best solvent for peptides extraction, to optimize the process conditions of extraction for maximal antifungal peptide recovery and to purify and characterize the peptide. Acidified organic solvent extracts of the larvae were tested against four fungi, namely, Aspergillus niger, Microsporum canis, Candida albicans and Blastomyces dermatitids. In vitro susceptibility results were observed by 'Colony Count Method', which was confirmed by two other methods, namely, 'Poisoned Agar Assay' and 'EUCAST Standard Method or Broth Micro Dilution Method'. In order to extract the maximal peptide, the process parameters of extraction has been statistically optimized by first executing One-Factor-At-a-Time (OFAT) strategy on three important parameters, namely blending time, initial solvent temperature and solid to solvent ratio, whereby the recorded responses were the extend of the growth of Aspergillus niger, which is the most susceptible fungus. This was followed by executing CCD design of experiments, to find the optimized extraction parameters and the interactions among them, for maximal peptides recovery. The crude sample was then purified using solid phase extraction (SPE) process and characterized by Biuret test, FTIR, Raman microscopy, UV-vis analysis, HPLC and the molecular weight was checked by SDS PAGE. Results showed that, out of four different tested, two extracts, namely, acidified isopropanolic and ethanolic were found to be active, although at varying degrees, towards the four fungi tested. The optimum peptides extraction conditions were 5 minutes homogenization time, 4°C initial solvent temperature and 1:3.5 solid to solvent ratio. The extraction carried out at the optimal conditions yield an extract which gave an absorbance of 3.99 at 230 nm and were able to inhibit the growth of Aspergillus niger up to 86.00%. Upon purification on SPE, the bioactive compound was found to be polar, based on the fact that this bioactive fraction was eluted during washing. Characterization experiments carried out on the bioactive samples confirms that it contains peptides. This is based on the Biuret test that showed a change of colour to violet in the presence of copper ions. FTIR analysis shows the presence of peak at 1632 cm-1 wavelength, which implies the presence of amide group I molecule. Raman microscopy analysis showed bands at 844, 1349 and 944 cm-1, indicating the presence of Tyr, Try and Gly respectively. Upon UV-vis analysis, it is confirmed that the sample contains peptides because of the presence of peak at 230 nm. UV-vis analysis also shows the presence of peaks between 260-280 nm, which implies that the samples have aromatic group containing molecules. Further, HPLC analysis confirmed the results obtained by Raman microscopy showing that the sample contains amino acids, Gly and Tyr, owing to the tested sample giving two peaks at the retention time of 7 and 8 minutes. This work is important because it may present the first initiative towards recovering antifungal peptides from low cost, easily found insect larvae, whereby it can be potentially developed as peptide-based drug to combat fungal infection. |
|---|---|
| Physical Description: | xix, 160 leaves : illustrations ; 30cm. |
| Bibliography: | Includes bibliographical references (leaves 134-152). |