Bioactive Betulin produced by marine Paecilomyces WE 3F

Article history: Received on: 30/12/2015 Revised on: 17/01/2016 Accepted on: 31/01/2016 Available online: 30/03/2016 Objective: Marine fungi play an important role in human and animal health, leading compounds to new drug discoveries and prospects for their bioactivity potential. Materials and Methods: Paecilomyces WE3-F was isolated from marine sediment (Red Sea, Shalateen, Egypt). Fungal isolate was screened for their antagonistic activity against four Gram-positive (Bacillus cereus, Lesteria monocytogenes, Micrococcus luteus and Staphylococcu aureus) and four Gram-negative (Aeromonas hydrophila, Flavobacteruim sp, Pseudomonas aeruginosa and Vibrio cholera,) pathogenic bacteria. Paecilomyces WE3-F was identified using 18S rRNA technology. Seven factors were chosen to be screened for bioactivity using the Placket Burman experimental design: sucrose, yeast extract, Na NO3, temperature, initial pH, inoculum size, and incubation period. Results: Among conditional factors, acidic pH and 1.5 ml inoculum size favored the bioactive metabolites. Furthermore, a number of solvents have been experimented for the extraction of the bioactive metabolite(s). Dichloromethane (DCM) crude extract from the fermentation broth of a marine Paecilomyces WE3-F showed the highest activity with averages of 26 and 24 mm against G-ve and G+ve, respectively. Under optimal culture conditions, the maximum extractable compound concentration in a 10-L culture medium reached 83.4 mg/L. Based on data obtained by thin layer chromatogram (TLC), gas chromatography mass spectrum (GC-MS) and Fourier Transform Infrared (FTIR) the major compound, betulin was structurally identified. Conclusions: The isolated marine Paecilomyces WE3-F, therefore, showed the ability to produce a betulin yield after optimal operating conditions for antibacterial potential.


INTRODUCTION
Betulin is a pentacyclic triterpenoid, manly found at the bark of several birch species (Alakurtti et al., 2006).The presence of reactive functional groups in betulin makes it an available and convenient starting material for synthesizing new biologically active compounds (Santos et al. 2009).In fact, the antibiotics era began in 1929 with the penicillin discovery by Fleming, who considered filamentous fungi as an important source of new drugs for therapeutic use (Yu and Keller 2005).Nowadays, this micromycetes by-product inspired scientists to re-explore new metabolites for biofactories, which synthesize desired pharmatheutical products for a healthy life.The production of terpenoids by endophytic fungi (65 sesquiterpenes, 45 diterpenes, 5 meroterpenes and 12 other terpenes) and their biological activities were reported by Jorgeane de Souza et al., (2011).Among these filamentous fungi, the ascomycete Paecilomyces species deserve special attention according to its reputation as a source of secondary metabolites without mycotoxins production (Mioso et al. 2015).P. variotii is found worldwide; it is commonly found in air, soil, and plant litter and was frequently recovered from aquatic environments where it probably resides as dormant conidia or ascospores (Marante et al., 2012;Silva et al., 2013).
In the continuous search for additional bioactive metabolites, a naturally occurring anacardic acid analogue was isolated from the same fungal strain showing cytotoxicity against human and antimicrobial activity against marine pathogens Escherichia coli DC2 and Streptococcus iniae FP5228 (Liu et al., 2012).Hyung et al., (2013) suggested that the viriditoxin (10) extract from marine P. variotii is effective for preventing and treating infections caused by fish pathogenic microorganisms, particularly Aeromonas hydrophila, A. sobria, Edwardsiella tarda, Clostridium botulinum, Mycobacterium marinum, Streptococcus iniae, Vibrio parahaemolyticus, V. cholerae, V. vulnificus and V. ichthyoenteri.These extracts that have been attributed to naturally occurring antibiotic in aquaculture could improve the safety of fish consumption, since it generates a lower environmental impact because of its low toxicity and rapid degradation (Mioso et al., 2014).
In the present study, the Placket Burmen experimental design was used to optimize the submerged culture conditions for the simultaneous production of bioactive betulin by Paecilomyces WE3-F.Meanwhile, the chemical characterization of betulin was carried out using TLC, GC-MS and FTIR.

Fungal Isolation
Marine fungi were originally isolated form Shalateen coast, Red Sea, Egypt.Briefly, sediment sample was collected in sterile plastic bags (APHA, 1998).The isolation process was carried out using the pour plate technique.One gm sediment in 30ml sterile sea water were applied on glucose peptone agar (GPA) and then incubated at 30 o C for 7 days at pH 7. Slants containing pure cultures were stored at 4°C until further use.The macroscopic examination of fungal hyphea was then observed.

Bacterial Pathogens
Four Gram-negative bacteria: Aeromonas hydrophila, Pseudomonas aeruginosa,, Flavobacterium sp., Vibrio cholera and four Gram-positive bacteria: Bacillus cereus, Lesteria monocytogenes; Staphylococcus aureus Micrococcus luteus were kindly provided by the Microbiology Lab, Marine Environment Department, National institute of oceanography and fishers, Egypt.Stock bacterial cultures were maintained on nutrient agar slants at 4 ˚C with monthly transfer.

Antibacterial Potential
Screening for the antibacterial potential was carried using the disk-diffusion assay method according to Kirby-Bauer (Bauer et al., 1966) to test the inhibition of marine fungi against the tested pathogens.GPA medium was used to support the secondary metabolite production.One ml of pre-cultured tested bacteria (OD 550 = 1.0) was mixed well using 25 ml of sterile and molten Muller-Hinton agar.Sterile discs of 6 mm diameter were immersed in the culture supernatant and placed on the seeded Muller-Hinton agar plates.All plates were incubated at 30oC for 24-48h.The inhibition zone diameters were recorded.

Identification of the Potent Isolate
Phylogenetical identification of the potent isolate was carried out using the manufacturer protocol of FavorPrep Fungi/ Yeast Genomic DNA Extraction Mini Kit (FAFYG 000).Gel documentation system (Geldoc-it, UVP, and England) was applied for data analysis using the Total lab analysis software, ww.totallab.com,(Ver.1.0.1).NCBI (http://www.ncbi.nlm.nih.gov/)software analysis was used for sequence alignment.Phylogenetic tree was constructed via ClustalW (http://www.ebi.ac.uk/Tools/msa/clustalw2/) software analysis.Isolated fungus was initially grown for 7 days on GPA medium in Petri dish.
A disc (5 mm) was cut from the margins of old culture of the agar plate culture and then transferred into the seed medium.The seed culture was grown in a 250 ml flask containing 100 ml GPA medium at 30 o C on a rotary shaker incubator at 120 rev/min for 7 days for further studies.

Effective Culture Media
Four culture media namely; potato dextrose, glucose peptone, czapek-dox and yeast-glucose liquid media, have been used to trace the antibacterial activity response.After adjusting pH to 7, the culture media were sterilized.Each medium was inoculated with 1 ml (1x107 spores/ml) then incubated at 30oC under shaking condition for 7 days.

Plackett-Burman Experimental Design
The effect of medium components on the bioactive compounds produced by marine fungus was studied by applying the Plackett Burman Experimental Design (Plackett and Burman, 1946;Greasham and Inamine, 1986).In this experiment, seven factors (medium components and culturing conditions) were determined in eight combinations organized according to the Plackett Burman matrix shown in Table 1.For each variable, a high level (+1) and a low level (-1) were tested (Rajendran et al., 2007).The factors tested were: Sucrose, NaNO 3 and yeast extract concentrations.Also, the pH, temperature inoculum size and incubation period of the tested fungus were also tested.The assays were performed in duplicate.
The main effect of each factor was determined using this equation: Exi = (ΣMi+ -ΣMi-)/N) Where Exi is the variable main effect; Mi+ and Mi-are the calculated results of the bioactivities (inhibition zone in mm).They are recorded by trial which contains positive and negative levels of independent variables (xi), respectively.N is the number of trials divided by 2. A main effect figure with a positive sign indicates that the positive level of this variable is nearer to optimum bio activity; while a negative sign indicates that the negative level of this variable is closer to optimum bioactivity.Using Microsoft Excel, statistical t-values for equal unpaired samples were calculated for the determination of variable significance (Al-Sarrani and El-Naggar, 2006).

Extraction of the Active Compound(S)
Four solvent systems namely: chloroform, dichloromethane, diethyl ether and ethyl acetate were individually applied for the extraction purpose.The organic layer was separated and concentrated under vacuum using rotary evaporator (STUART, RE300DB) to obtain the oily crude substance.Ten liters of culture filtrate of marine fungus were extracted from the supernatant using 1:1 (v/v) supernatant: dichlormethane.The overall crude extract amounted to 834 mg.The crude extract was then tested using the disc diffusion assay method.

Thin layer chromatography
The selected dichloromethane crude extract was fractionated using the silica gel thin layer chromatography sheet (60GF254 Merck).Then, the crude was applied as a spot and left to complete separation.The plates stood for solvent evaporation and the R f of the result spot was recorded.The collected samples were evaporated, weighed and dissolved in dichlormethane to be applied on pathogens inoculated plates.The bioactivity of the collected samples was checked for the eight pathogens using the disc diffusion assay.

Gas Chromatography-Mass Spectrum analysis
Oily crude was dissolved in 10 ml of dichlormethane and subjected to chromatographic analysis.The active dichlormethane fraction that was subjected to Gas chromatograph-mass spectrometer coupling (GC/MS) measurements were performed on an Agilent Technologies 7890A GC System with a flame ionization detector, a 5975C inert XL MSD Triple-Axis Mass Detector and Agilent 19,091S-433 Trace Analysis column.GCconditions: 1 µL of sample was injected with an evaporation temperature of 250 °C, 1.8 bar, 2.5 mL/min, split 20:1.He, the carrier gas temperature was gradient 50 °C/1 min, 40 °C/min gradient 300 °C/min, 300 °C/5 min.The components were identified by comparing their retention times to those of authentic samples, as well as by comparing their mass spectra with those of Wiley 275 Library (Wiley, 2006).Quantitative data were obtained by the peak normalization technique using integrated FID response.

Fourier Transform Infrared analysis
Dichloromethane crude was subjected to FTIRspectroscope (Perkin Elmer, USA) in the mid IR region of 400-4000 cm -1 with 16 scan speed using KBr discs.

RESULTS AND DISCUSSION
Paecilomyces sp.isolated from a broad range of habitats drives the diversification of new high-value-added secondary metabolites that play an important role in a healthy life, leading new drug discoveries, developments of new trends, and prospects for the bioactivity potential of this ascomycete (Mioso et al., 2015)

MARINE ACTIVE ISOLATE
After 7 days of incubation, marine Paecilomyces WE3-F was selected according to its highest antibacterial activities, where, the maximum activity recorded was 25 mm against V. cholera as Gram positive and 27 mm against S. aureus as Gram negative; whereas, lower activity recorded for both B. cereus and L. monocytogenes at 10 mm (Table 2).Compared with our results, cell free culture from Paecilomyces fumosoroseus obtained after 21 days of incubation exhibited highest activity for pathogenic bacteria including: Escherichia coli (32 mm), Bacillus subtilis (18 mm) and Salmonella typhi (12 mm); whereas, it was lower for Pseudomonas aeruginosa (10 mm), and showed no activity for Klebsiella pneumoniae (Gulwani et al., 2015).Also, different Pacelomyces sp.cultivated on PDA for 5 days, exhibited antibacterial activities against B. subtilis, S. aureus with averages of inhibition zone 27 and 29 mm, respectively (Lee et al., 2005).
In our present study, the marine potent isolate was analyzed for its 18S rRNA gene, and the phylogenetic relationship showed that this strain is very close to the strain type, Paecilomyces WE3-F (Figure 1).

EFFECTIVE MEDIA FOR BIOACTIVITY
Averages of inhibition zone of Paecilomyces WE3-F using four culture media was represented in Table 3 a & b.Using Czapek-dox medium, containing yeast and sucrose as main N and C sources, the highest activities were recorded at 23 mm and 19.24 mm against G-ve and G+ve, respectively; whereas, lower activities were represented using potato dextrose and yeast glucose medium.Two different liquid culture using P. fumosoroseus were tested for biocontrol efficacy using basal salts medium (Sandoval-Coronado et al., 2001).Another ten different culture media were used to investigate a mycelia growth of P. fumosoroseus, where, only two media were the favorable carbon and nitrogen source for maximal growth (Shim et al., 2003).Recently, the growth of P. hepiali in various agar media was studied, where, the addition of peptone improved mycelial growth and the most favorable carbon sources were mannose, fructose and glucose (Chioza, and Ohga, 2013).
Our study revealed that Czapek-dox medium containing in g/l: sucrose 20; NaNO 3 2; yeast 2, were further used within an experimental design to elucidate the maximum activity of Paecilomyces WE3-F.

ELUCIDATION OF MEDIUM COMPONENTS FOR THE BIOACTIVITY
Optimization of the components of the selected Czapekdox broth medium for the maximum activity using Paecilomyces WE3-F was determined.The nutrient medium factors and cultivation conditions were screened by applying the Plackett-Burmann design as described in the materials and methods section.Table 4 shows that trial 5 yielded the highest activity of Paecilomyces WE3-F at 19.9 mm which was obtained by incubating 1.5 ml (2.3 x 10 7 spores/ml) of the fungus for 5 days in 100 ml medium containing in g/l: sucrose 10, NaNO 3 2.5 and yeast extract 3, at incubation temperature of 25°C and pH 5.These results revealed that the degree of significance of pH and inoculum size was at 95 and 80%, respectively.It also showed that the decrease of pH and the increase of inoculum size caused an increase in activities using marine Paecilomyces WE3-F (at the 5 and 20 % level of significance) (Figure 2).Using response surface methodology to optimize the extract process of mycelial polysaccharides from P. hepiali, as well, test antioxidant activities were studied by Yu et al (2011).
In addition, the best growth for P. hepiali in an optimized media was obtained at 25°C in 14 days with initial pH range from 6 to 8 (Chioza, and Ohga, 2013).Giraldo et al. (2012) observed that P. variotii is able to produce extracellular invertases when grown for 96 h, where, the optimal temperature and pH values were 60°C and 4•0-4•5, respectively.Statistical method involves changing one independent variable was carried out for medium optimization condition for Inonotus obliquus yield of betulin, where, optimal pH was at 6.0 and optimum temperature was at 25 o C (Bai et al., 2012).

DETERMINATION OF THE APPROPRIATE SOLVENT(S) FOR EXTRACTION OF THE ACTIVE AGENT
The results showed that dichloromethane solvent was selected for the extraction procedures where the yielded extract obtaining showed the highest average zone of inhibition; 26 and 24 mm against Gram -ve and +ve, respectively (Figure 3).However, the other solvent showed low or no antimicrobial activity against the eight pathogens (Figure 4).Compared with our results, the extracts from P. lilacinus were evaluated for antimicrobial activity by disc diffusion method showed activity for Gram-positive S. aureus and L. monocytogenes at average inhibition zone 20.76 mm, even while, Gram-negative bacteria were not affected by the tested compounds (Teles et al., 2013a).The antimicrobial activity of the methanol crude extract from marine-derived Paecilomyces sp. was determined for MRSA, Escherichia coli, A. hydrophila and C. albicans at 18, 6, 9 and 7 mm, respectively (Liu et al., 2011).Ethyl acetate extract of P. variotii FEL24 presented antimicrobial activities ranged from 19 to 35 mm (Silva et al, 2011).The active ethyl acetate extract of P. lilacinus showed antibacterial activity for some pathogenic bacteria by agar diffusion assay: 19, 19.3 and 16 mm against Ps.syringae, R.   solanacearum and E. coli; whereas, there was no activity against X.vesicatoria, B. cereus B. subtilis and S. aureus (Srivastava and Anandrao, 2015).

PHYSICOCHEMICAL CHARACTERIZATION OF THE CRUDE EXTRACT
Paecilomyces genus includes many species capable to produce secondary metabolites which belongs to different chemical groups with wide biological activity such as paecilosetin (Lang et al., 2005), paecilaminol (Ui et al., 2006), paecilodepsipeptide A (Isaka et al., 2007), paecilin A andB (Guo et al., 2007).Using silica gel TLC plate, dichloromethane crude extract was run as concentrated spots (R f = 0.90) then scratched to estimate its bioactivity for the eight tested pathogens by disc diffusion assay.The extracts from P. lilacinusto assayed were prepared by dissolution on a suitable solvent and applied in readymade TLC plates that developed a yellow color in the plates (Teles and Takahashi, 2013b).The extract of P. variotii FEL32 strain submitted to TLC yielded two spots of R f 0.48 and 0.70 (Silva et al, 2011).Spectroscopic analysis of the separated spot .showed a major sharp peak observed at acquisition time 29 min with the highest count percentage 1x10 2 on gas-chromatography.This major peak was identified as betulin with the aid of mass spectrum library (Figure 5A & B).More recently, a new strain of P. variotii was isolated from a marine habitat showed 28 structural groups of volatile organic compounds identified by GC-MS from liquid fermented biomass, where, active terpenoids were represented (Marante et al., 2012).Moreover, mass spectrum of viriditoxin obtained from marine P. variotii was studied by Silva et al. (2013).
FT-IR spectrum of betulin showed the absorption band frequency at 3448 cm -1 with the presence of O-H stretch.In the area of 3059 cm -1 appears derivates from C-H stretch for alkane in CH 2 group and at 1765 cm -1 appears a very intensive absorption characteristic ribbon of carbonyl group (C=O).The1598 cm -1 band can be assigned to C=C stretching and CH 2 bending vibrations of the methyl and of the CH 2 groups in the rings.The band at 1006 cm -1 can be assigned to C-O stretching vibrations in the CH 2 OH group.A strong absorption band at 713 cm -1 indicated the presence of wagging vibrations in the terminal group aromatic C-H (Figure 6).The FT-IR spectrum of bioactive exopolysaccharide from a native Paecilomyces sp.showed characteristic bands at different wave lengths (Naturforsch, 2011).In conclusion, work reports the isolation of a marine Paecilomyces WE3-F showing the ability to produce betulin with antibacterial potential.
Using Plackette Burman experimental design, it was possible to determine optimal operating conditions to obtain a high betulin yield.This metabolite could be used in further investigation as a potential candidate for analytical purposes and /or further biotechnological applications.

Fig. 4 :
Fig. 4: Antibacterial activity of dichloromethane extract against Gram-positive and Gram-negative tested bacteria using disc-diffusion method.

Fig. 5 :
Fig. 5: Gas Chromatography-Mass Spectra showing the major peak in GC analysis (A) and identified compound "betulin" in Mass Spectral analysis (B) .

Table 3 :
Bioactivity of Paecilomyces WE3-F at different culture media for Gram -ve and Gram +ve bacteria.

Table 4 :
Degree of positive or negative effect of independent variables on bioactivity of Paecilomyces WE3-F according to levels in the Plackett Burman experiments.