Characterization and bioassay of penicillin (Penicillium Chrysogenum) produced from oranges (Citrus Sinensis) collected from Na'ibawa orange market, Kano State, Nigeria

ABSTRACT

This study aimed at producing penicillin from the rotten, fresh and preserved rotten oranges. The samples were prepared by extracting the orange juice and their pHs adjusted to pH 6.5 using 1% calcium hydroxide; then sterilized for fifteen minutes using an autoclave at 121°C. The samples were then inoculated with Penicillium chrysogenum and kept for fifteen days. The inoculated samples were analysed using Thin Layer Chromatography (TLC), Fourier Transform Infrared Spectroscopy (FTIR), and Gas Chromatography Mass Spectrometry (GC-MS). The FTIR spectra of rotten and fresh oranges were similar to that of the Benzathine benzylepenicillin standard. The spectra indicated a broad band at 3342 cm-1, representing bonded –OH stretching vibration and the –NH stretching of a protein. The peaks appearing in the 2977 cm-1 and 2836 cm-1 regions may be attributed to the asymmetric and symmetric stretching vibration of -CH2, respectively. The strong adsorption band at 1659 cm-1 represents -C=O stretching vibration and NH deformation (amide I). The peak at 1566 cm-1 was assigned to a motion combining both –NH bending (amide II) and –CN stretching vibration of the protein. The typical amide III band, which represents -COO- anions, appeared at 1380 cm-1. The weak peak at 1398 cm-1 was induced by the -C-N stretching vibration of amine groups. The strong adsorption peaks 1249 cm-1 and 1026 cm-1 are indicators of –SO3 groups and the -C-N stretching vibration of amine groups, respectively. The moderate peak at 888 cm-1 may be attributed to aromatic –CH stretching vibration. The fingerprint zone of the spectra, ranging from 650 cm-1 to 800 cm-1, represents phosphate or sulphur functional groups. Bioactive compounds were identified from the penicillin produced from rotten and fresh oranges using GC-MS analysis. The most prevalent were; cis-13-octadecenoic acid, methyl ester, hexadecanoic acid, pentyl ester, 9-octadecenoic acid (Z)-,2,3-dihydroxypropylester, 9-Octadecenoic acid (Z)-,2-hydroxy-1-(hydroxyl methyl)ethyl ester and 4-Oxatricyclo [20.8.0.0(7,16)] triaconta-1(20),7(16)-diene, at a retention times of; 13.6 min, 16.05 min, 21.90 min, 21.94 min and 30.86 min respectively. The highest zone of inhibitions observed before recovery due to Escherichia coli and Streptococcus for the rotten and fresh oranges were; 22.4 mm and 18.24 mm respectively; while inhibition was not observed for the preserved rotten oranges. However, after recovery, the highest zone of inhibitions observed due to E.coli, Methicillin-resistant Staphylococcus aureus (MRSA), Meticillinic-Sencitive Staphylococcus aureus (MSSA) and Klepsiella were 26 mm for the standard, 8 mm for the fresh orange, 32 mm for the fresh orange, and 34 mm for the rotten orange respectively. The highest yield (400 mg/cm3) of the produced penicillin was the recorded for the rotten orange before optimization. The yield remains the same after optimization. The study demonstrated that fresh and rotten oranges are viable substrates for the fermentation media to be used in cultivating fungus for the production of natural penicillin antibiotics.