Determination of metabolites products by Penicillium expansum and evaluating antimicobial activity

The objectives of this study were to analyze the secondary metabolites of Penicillium expansum and evaluate antibacterial activity. Twenty eight bioactive compounds were identified in the methanolic extract of P. expansum. The identification of bioactive chemical compounds is based on the peak area, retention time molecular weight and molecular formula. Gas chromatography mass spectrometry (GC/MS) analysis of P. expansum revealed the existence of the Levoglucosenone, Edulan ll, 4[Dichloromethyl]-2-[[2-[1-methyl-2-pyrrolidinyl]ethyl]amino-6-trichloro, 1,2-Cyclopentanedione, Ethanethiol, 2-(5-(4-methyl-2-pyridyloxy)pentyl)amino-hydrogen su, Imidazole,2-amino-5-[(2carboxy)vinyl], D-Glucose,6-O-α-D-galactopyranosyl, Eicosanoic acid , phenylmethyl ester, Dodecanoic acid, 3hydroxyl, DL-Leucine,N-glycyl, Cyclohexene,1,5,5-trimethyl-6-acetylmethyl, 1,2-Nonadecanediol, Bicyclo[2.2.1]heptane-2-carboxylic acid isobutyl-amide, 6-Acetyl-ß-d-mannose, α-DGlucopyranoside,O-α-D-glucopyranosyl-(1.fwdarw.3)-ß-D-fruc, propanedioic acid, amino-diethyl ester, 4H-Pyran-4-one,2,3-dihydro-3,5-dihydroxy-6-methyl, valeric acid, dodecyl ester, deoxyspergualin, IGala-I-ido-octonic lactone, 5-Hydroxymethylfurfural, paromomycin, 16-Nitrobicyclo[10.4.0]hexadecane1-ol-13-one, cis-Vaccenic acid, 2-Bromotetradecanoic acid, phthalic acid, butyl undecyl ester, picrotoxin, D-Fructose and diethyl mercaptal. The FTIR analysis of P. expansum proved the presence of aliphatic fluoro compounds, tetiary amine, C-N stretch and methylene-CH. asym which shows major peaks at 1028.06, 1151.50 and 2852.72, respectively. Methanolic extract of bioactive compounds of P. expansum were assayed for in vitro antibacterial activity against Proteus mirabilis, Pseudomonas aerogenosa, Escherichia coli, Staphylococcus aureus and Klebsiella pneumonia by using the diffusion method in agar. The zones of inhibition were compared with different standard antibiotics. S. aureus has maximum zone formation (7.01±0.141) mm.


INTRODUCTION
The genus Penicillium is known worldwide for the production of secondary metabolites (Pitt and Hocking, 1997).Penicillium expansum has been demonstrated to produce extracellular enzymes of commercial value, including the pectinases, utilized in fruit juice industry during the stage of pulp maceration, juice liquefaction or depectinization (Rosenberger et al., 1991;Baracat-Pereira et al., 1989), Patulin, a mutagenic, immunoitoxic and neurotoxic mycotoxin, particularly unacceptable to apple juice industry (Bracket and Marth, 1979).Most Penicillium species are considered ubiquitous, opportunistic saprophytes.Blue mold decay caused by P. expansum link is the most important postharvest disease of apple worldwide (Pierson et al., 1971).The genus Penicillium is subdivided into four subgenera (Aspergilloides, Penicillium, Biverticillium and Furcatum), determined by the number of branch points between phialide and stipe, down the main axis of the penicillus and others characters, like ratio of metula length to phialide, length and colony diameter on G25N and when the number of branch points is the same (Grassin and Fauquembergue, 1996).
Nutritionally, they are supremely undemanding being able to grow in almost any environment with a sprinkling of mineral salts, any but with the most complex forms of organic carbon, and a wide range of physical-chemical environments, temperature, pH and redox potential.The taxonomy of this genus is hard as its classification is based mainly on conidiophore and conidia structure, although the colony diameter after incubation under standardized conditions has greater importance for classification (Amiri and Bompeix, 2005;Morales et al., 2008;Hameed et al., 2015a).P. expansum is one of most important fungal pathogen of stored pome fruits and responsible for 50% of losses in all pome fruit and pear (Mattheis and Roberts, 1992;Andersen et al., 2004;Murphy et al., 2006;Hameed et al., 2015b).

MATERIALS AND METHODS
Collection, extraction and determination of metabolites P. expansum was isolated from dried fruit.After the species were identified by the identification key, spores were grown in a liquid culture of potato dextrose broth (PDB) and incubated at 25°C in a shaker for 16 days at 130 rpm (Usha and Masilamani, 2013).The metabolites were determined and extracted for GC analysis using the method of Jasim et al. (2015).The extraction was performed by adding 25 ml methanol to 100 ml liquid culture in an Erlenmeyer flask after the infiltration of the culture.The mixture was incubated at 4°C for 10 min and then shook for 10 min at 130 rpm.Metabolites was separated from the liquid culture and evaporated to dryness with a rotary evaporator at 45°C.The residue was dissolved in 1 ml methanol, filtered through a 0.2 μm syringe filter, and stored at 4°C for 24 h before being used for GC-MS.

Gas chromatography-mass spectrometry (GC-MS)
Bioactive compound were examined for the chemical composition using GC-MS (Agilent 789N) equipped with a DB-5MS column (30 m×0.25 mm i.d., 0.25 um film thickness, J&W Scientific, Folsom, CA).The oven temperature was programmed (Imad et al., 2014a;Hussein et al., 2015).Helium was used as the carrier gas at the rate of 1.0 ml/min.Effluent of the GC column was introduced directly into the source of the MS via a transfer line (250°C).Ionization voltage was 70 eV and ion source temperature was 230°C.Scan range was 41 to 450 amu.The constituents were identified after being compared with available data in the GC-MS library in the literatures of Kareem et al. (2015) and Imad et al. (2014b).

Fourier transform infrared spectrophotometer (FTIR)
The sample was run at infrared region between 400 and 4000 nm.The powdered sample of the P. expansum specimen was treated for fourier transform infrared spectroscopy (Shimadzu, IR Affinity 1, Japan) (Imad et al., 2014c).

Determination of antibacterial activity of crude fraction of P. expansum compounds.
The test pathogens (E.coli, Pseudomonas aeruginosa, Klebsiella pneumoniae and Staphylococcus aureus) were swabbed in Muller Hinton agar plates.50μl of fungal extracts was loaded on the bored wells.The wells were bored in 0.5 cm in diameter.The plates were incubated at 37°C for 24 h and examined.After the incubation the diameter of inhibition zones around the discs was measured.

Statistical analysis
Data were analyzed using analysis of variance (ANOVA), and differences among the means were determined for significance at P < 0.05 using Duncan's multiple range test (by SPSS software)Version 9.1 (Mohammed and Imad, 2013)

Fungus identification and secondary metabolites production
The fungi were isolated by serial dilution method.Morphological, microscopical and microscopical characteristics of fungal strains were determined using specific media light and compound microscope (Figures 1  and 2).After fermentation, the secondary metabolites were produced by isolated microorganisms.

Identify the secondary metabolites from the methanolic crude extract of P. expansum by (GC-MS)
Gas chromatography and mass spectroscopy analysis of compounds was carried out in methanolic extract of P. expansum, shown in Table 1.The GC-MS chromatogram of the twenty eight peaks of the compounds detected was shown in Figure 2. Chromatogram GC-MS analysis of the   methanol extract of P. expansum showed the presence of twenty eight major peaks, and the components corresponding to the peaks were determined as follows.The first set up peak was determined to be levoglucosenone, Edulan ll, (Figure 3).

Identify the secondary metabolites from the methanolic crude extract of P. expansum by (FTIR)
Fourier-transform infrared analysis of dry methanolic extract of P. expansum proved the presence of aliphatic fluoro compounds, tetiary amine, C-N stretch and methylene-CH.asym which shows major peaks at 1028.06, 1151.50 and 2852.72,respectively (Table 2 and Figure 31).

Conclusion
This study showed that P. expansum produce many important secondary metabolites with high biological activities.Based on the significance of employing bioactive compounds in pharmacy to produce drugs for the treatment of many diseases, the purification of compounds produced by P. expansum can be useful.

Table 1 .
Bioactive chemical compounds identified in methanolic extract of P. expansum.

Table 3 .
Antibacterial activity of bioactive compounds of Penicillium expansum against bacterial strains.