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Production, purification and characterization of an antimicrobial compound from marine Streptomyces coeruleorubidus BTSS-301
j o u r n a l o f p h a r m a c y r e s e a r c h 7 ( 2 0 1 3 ) 3 9 7 e4 0 3
Available online at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/jopr
Original Article
Production, purification and characterization of an antimicrobial compound from marine Streptomyces coeruleorubidus BTSS-301 Siva Kumar Kandula a,*, Ramana Terli b a
Research Scholar, Department of Biotechnology, College of Science and Technology, Andhra University, Visakhapatnam 530003, Andhra Pradesh, India b Dean of Life Sciences, GITAM Institute of Science, GITAM University, Visakhapatnam 530045, Andhra Pradesh, India
article info
abstract
Article history:
Background: An indigenous marine Streptomyces coeruleorubidus BTSS-301 has been isolated
Received 14 March 2013
from marine sediment sample near Visakhapatnam coast at a depth of 30 m. It exhibited
Accepted 27 April 2013
broad spectrum of antimicrobial activity. However, for maximum production of a specific
Available online 2 June 2013
bioactive compound from indigenous isolates medium formulation is an essential step. Methods: Optimization experiments for various carbon and nitrogen sources and cultural
Keywords:
parameters were performed by shake flask culture method. The active compound in cul-
Antimicrobial compound
ture filtrate was extracted into ethyl acetate and then purified by silica gel column chro-
Biomass
matography. The purified active compound was characterized by spectroscopy studies.
Medium optimization
The minimum inhibitory concentration was determined by broth dilution method.
Minimum inhibitory concentration
Results & discussion: Optimization studies revealed that the highest antibiotic production
Streptomyces coeruleorubidus
was obtained when glucose at 10.0 g/l and NH4NO3 at 2.5 g/l were used as carbon and nitrogen sources respectively. Cultural parameters revealed that 96 h of incubation at 30 C at 180 rpm with pH 7.2 of the production medium exhibited maximum yield. The purified active compound is identified as N-ethyl-2-(2-(3-hydroxybutyl) phenoxy) acetamide. Conclusion: The antimicrobial compound from S. coeruleorubidus BTSS-301 could be a candidate in the generation of new antimicrobial agents from marine Streptomyces. Copyright ª 2013, JPR Solutions; Published by Reed Elsevier India Pvt. Ltd. All rights reserved.
1.
Introduction
Streptomyces are the most economically and biotechnologically valuable prokaryotes. They are responsible for the production of about half of the discovered bioactive secondary metabolites such as antibiotics, antitumor agents and immunosuppressive agents.1 The identification of new
compounds from terrestrial Streptomyces has gradually decreased and the re-isolation of existing metabolites has increased.2 Thus, marine habitats were screened for novel bioactive secondary metabolites. As marine environmental conditions are extremely different from terrestrial ones, it is assumed that marine Streptomyces might produce different types of bioactive secondary metabolites.3,4 The success of
* Corresponding author. Tel.: þ91 9912286860; fax: þ91 (0) 891 2734821. E-mail address: [email protected] (S.K. Kandula). 0974-6943/$ e see front matter Copyright ª 2013, JPR Solutions; Published by Reed Elsevier India Pvt. Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jopr.2013.04.047
398
j o u r n a l o f p h a r m a c y r e s e a r c h 7 ( 2 0 1 3 ) 3 9 7 e4 0 3
screening programs for antibiotic production is heavily dependent on the identification of isolates to the correct taxa. However for indigenous isolates it is essential to grow in a diverse range of production media including the use of formulations which mimic conditions in the environment in the case of strains from marine habitats.5 Medium formulation is an essential stage for the successful production of a specific bioactive compound. The media used for submerged cultivation of Streptomyces have a dramatic impact on the expression of secondary metabolite gene clusters.6 The antibiotic production is highly based on the carbon/nitrogen ratio in the medium. Medium with a high content of both carbon and nitrogen source (3.5:1, C/N ratio) permits optimal growth of nearly all actinomycetes strains.7 Several clinically useful compounds were reported from Streptomyces coeruleorubidus. These include pacidamycins, antifungal metabolites, and indispensable anticancer drugs such as, doxorubicin and epirubicin.8 The present study was undertaken to examine the effect of different nutrients and cultural conditions on antimicrobial compound production and to purify extra cellular compound from the indigenous marine isolate S. coeruleorubidus BTSS-301 and to determine the structure of the purified compound.
2.
Methods
2.1.
Microorganism
The indigenous organism designated as BTSS-301, was isolated from a marine sediment sample collected from Bay of Bengal near Visakhapatnam coast at a depth of 30 m. Morphological, cultural and physiological characteristics of the strain were studied using the International Streptomyces Project (ISP) media recommended by Shirling and Gottlieb9 and was taxonomically characterized by using Polyphasic approach. The isolate has been identified as S. coeruleorubidus10 (Data published).
2.2.
2.4.
Basal medium
To determine the optimal nutritional and cultural conditions for growth and antimicrobial activity, Pridham and Gottlieb’s11 inorganic salt medium was used as the production medium base.
2.5. Optimization of media and cultural conditions for antimicrobial compound production The effect of various carbon sources, glucose concentration, organic nitrogen sources, inorganic nitrogen sources, NH4NO3 concentration, metal ions and cultural conditions were optimized by using shake flask culture method. The biomass from the culture filtrate was separated by means of centrifugation. It was transferred to pre weighed dry Whatman No. 1 filter paper. The filter paper along with the biomass was dried in a hot air oven at 80 C for 18e24 h to reach a fixed weight. Growth was expressed in terms of dry weight as mg/ml culture medium.
2.6.
Fermentation
The S. coeruleorubidus BTSS-301inoculum was introduced aseptically into sterile flasks containing ingredients (g/l) glucose, 10; NH4NO3, 2.5; K2HPO4, 2.0; MgSO4.7H2O, 1.0; and trace salt solutions9 1.0 ml, with pH of the medium 7.2. The flasks were incubated for 96 h at 30 C at 180 rpm. The culture filtrate was then separated by centrifugation at 3000 rpm for 15 min.
2.7. Extraction and purification by column chromatography
Test microorganisms
The following microorganisms procured from IMTECH, Chandigarh, India were used during the investigation as test microorganisms. Staphylococcus aureus (MTCC 3160), Bacillus subtilis (MTCC 441), Bacillus cereus (MTCC 430), Pseudomonas aeruginosa (MTCC 424), Escherichia coli (MTCC 443), Proteus vulgaris (MTCC 426), Saccharomyces cerevisiae (MTCC 170), Candida albicans (MTCC 227), Aspergillus niger (MTCC 961), and Aspergillus flavus (MTCC 3396).
2.3.
suspended in saline solution. 5 ml of this suspension was used as inoculum for the optimization experiments by shake flask culture.
Inoculum preparation
Seed medium composed of (g/l) soluble starch 25; Ammonium sulfate, 5; NaCl, 5; CaCO3, 5 with pH adjusted to 7.0 was used for the seed production. For the seed growth, mycelium from a seven day old, well-sporulated slant of the culture was inoculated into 200 ml of seed medium and grown at 28 C with 120 rpm on a shaker incubator for 48 h. Then culture was centrifuged at 3000 rpm for 10 min to separate the cells from the broth. The cell pellet was washed thoroughly and
The culture filtrate (12 l) was extracted twice with ethyl acetate and the pooled solvent extracts were evaporated to dryness under reduced pressure to yield a crude residue. The purification of the antimicrobial compound was carried by using silica gel column (2.5 25) chromatography. Silica gel of 100e200 mm particle size was used for packing the column. Chloroform and methanol (7:3, v/v) were used as an eluting solvent. 5 g of crude extract to be fractioned was dissolved in 50 ml of methanol and passed through the silica gel column keeping the flow rate at 0.2 ml/min; thirty fractions were collected (5 ml each) and tested for their antimicrobial activities. The purity of the active fraction was determined by Waters Reverse Phase HPLC, Spherisorb 5 mm ODS 2 (C18) column with solvent system methanol and water 70:30 (v/v) at 2500 psi in isocratic mode. The operating flow rate was 1.0 ml/min. The solubility pattern of the compound was determined in various polar and non-polar solvents. The melting point of the compound was determined by FishereJohns melting point apparatus.
j o u r n a l o f p h a r m a c y r e s e a r c h 7 ( 2 0 1 3 ) 3 9 7 e4 0 3
2.8.
Spectroscopic analysis
The UV absorption spectrum of the compound was determined by Shimadzu UV 1800 spectrophotometer. The Infrared (IR) spectrum of the purified antimicrobial compound was recorded using Bruker Alpha FT-IR spectroscopy. The resulting data generated was viewed with the help of OPUS v6.5 software. NMR spectrum of the compound was determined by using an AMX-400 spectrometer (Bruker, Germany) 1 H data was obtained at 399.7 MHz and 13C was at 100.5 MHz using chloroform-d as solvent and trimethylsilane as internal reference.
2.9.
Minimum inhibitory concentration (MIC)
The minimum inhibitory concentration has been determined by broth dilution method.12 The media used were nutrient broth for bacteria and Czapek Dox broth for fungi.
3.
Results
3.1.
Effect of carbon source
3.2.
Effect of nitrogen source
Among the various inorganic nitrogen sources, the maximum metabolite production was achieved with
Table 1 e Effect of different carbon sources on growth and metabolite production. Carbon sources 1% (w/v) D-Glucose D-Fructose D-Galactose D-Xylose Arabinose Glycerol Starch Maltose Lactose Sucrose Meso-inositol Mannitol
NH4NO3 (192 mg/ml) with biomass of 3.8 mg/ml. Among the organic nitrogen sources, the high level of metabolite yield was obtained with soyabean meal (Table 2). Further, the concentration of 2.5 g/l of NH4NO3 (Fig. 1) greatly influenced the antimicrobial compound production with maximum yield and biomass accretion of 3.3 mg/ml. Moreover the yield was reduced with increase and decrease of NH4NO3 concentration.
3.3.
Growth in dry weight (mg/ml)
Antibiotic concentration (mg/ml)
3.0 0.8 0.8 0.4 0.5 2.4 2.2 0.8 0.7 0.8 0.6 0.3
160 50 58 38 44 112 120 20 18 50 48 35
Effect of amino acids
Different amino acids were added to the improved production medium containing glucose and NH4NO3 as carbon and nitrogen sources. Out of 13 amino acids, only arginine, glutamate, asparagine, aspartate, tryptophan and histidine favored the growth and metabolite production (Table 3). Among them, arginine, glutamate and tryptophan promoted the maximum biomass accumulation (2.6 mg/ml) than the other amino acids. The remaining amino acids yielded relatively less amount of antibiotic.
3.4.
The optimization of the metabolite production was carried out in batch cultures. The isolate BTSS-301 was cultivated in basal medium supplemented with different carbon sources, and their effect on growth and antimicrobial activity was studied (Table 1). The isolate grow in all the test carbon sources. Maximum metabolite production was obtained with glucose (160 mg/ml) followed by glycerol (120 mg/ml) and starch (112 mg/ ml) and the biomass obtained was also highest with glucose (3 mg/ml) than that of glycerol and starch. The effect of different concentrations of glucose (Fig. 1) on growth and production showed that the antibiotic titer was highest with 10 g/l glucose concentration with biomass of 3.6 mg/ml.
399
Effect of metal ions
The maximum biomass (3.6 mg/ml) and metabolite production was favored at 2.0 g/l concentration of K2HPO4 (Fig. 1). Similarly the effect of different concentrations of MgSO4.7H2O on growth and metabolite yield was also studied. The results indicate that the concentration of both the metal ions strongly influence the antibiotic production. The concentration of 1.0 g/l MgSO4.7H2O promoted the maximum growth (3.2 mg/ ml) and antimicrobial compound production (Fig. 1).
3.5.
Effect of cultural parameters
In addition to culture media, cultural conditions strongly influence the antimicrobial compound production. The effect of cultural conditions on growth and production by the isolate BTSS-301 has been studied in detail. Maximum antibiotic yield was obtained at 30 C with biomass of 3.6 mg/ml (Fig. 1). The increase of incubation temperature from 20 C to 30 C increased the growth of biomass and the production of metabolite respectively. However, the yield decreased consistently with the cell mass by increasing the growth temperature range from 35 to 50 C. Even though biomass was deposited at 45e50 C, the antibiotic yield was negligible. The maximum antibiotic yield was obtained at pH 7.2 with a biomass of 2.8 mg/ml (Fig. 1). The growth and antibiotic production by the isolate BTSS-301 was monitored over a period of 120 h. The antibiotic production occurred in a growth phase dependent manner and the highest yield was obtained in the late exponential phase and the stationery phase. The maximum yield was obtained at 96 h incubation period with biomass of 3.9 mg/ml (Fig. 1). The agitation provides greater aeration to the culture and also creates conditions for greater availability of nutrients to cells. The highest metabolite yield was obtained at 180 rpm with biomass of 3.2 mg/ml (Fig. 1). Further increase in the agitation speed demonstrated rapid decrease in yield along with biomass.
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Fig. 1 e Effect of nutrients, metal ions and cultural conditions on biomass and metabolite production.
3.6.
Fermentation, extraction and purification
The fermentation process was carried out for 96 h at 30 C. After incubation period, the culture supernatant was separated by centrifugation at 3000 rpm for 15 min. the culture filtrate was extracted twice with ethyl acetate (1:2, v/v) and the organic layer was evaporated to dryness under reduced pressure to give yellow colored precipitate. 5 g of the precipitate in 50 ml of methanol was chromatographed on silica gel column using solvent system chloroform and methanol (7:3, v/v). A total of 30 fractions of 5 ml each were collected. Among all the fractions tested for antimicrobial activity, active fractions were ranged between fraction no.11e23. Further
purification was carried out by silica gel column chromatography to remove minor impurities and unsolicited metabolites. A total of 20 minor fractions of 2 ml each were collected. All of them were subjected to TLC analysis and fractions with similar Rf (0.69) values were pooled together. Finally three major fractions were obtained IIIa (232 mg), IIIb (23 mg) and IIIc (10 mg). Out of these three fractions, fraction IIIa exhibited highest antimicrobial activity when compared with the remaining two fractions. The purity of the active fraction was analyzed by reverse phase HPLC, confirming the 95% purity of the compound. The compound was obtained in form of a crystalline yellow colored solid material. It was soluble in DMSO, methanol,
j o u r n a l o f p h a r m a c y r e s e a r c h 7 ( 2 0 1 3 ) 3 9 7 e4 0 3
Table 2 e Effect of different nitrogen sources on growth and metabolite production. Nitrogen sources
Growth in dry weight (mg/ml)
Inorganic nitrogen sources (0.2% w/v) Ammonium citrate 1.6 Ammonium nitrate 3.8 Ammonium sulfate 1.9 Potassium nitrate 1.5 Sodium nitrate 1.8 Organic nitrogen sources (0.2% w/v) Soyabean meal 3.1 Peptone 1.5 Beef extract 1.9 Yeast extract 2.4 Tryptone 1.8
Antibiotic concentration (mg/ml) 122 192 155 98 138 192 89 142 160 110
ethanol, acetone, ethyl acetate, chloroform, and diethyl ether but insoluble in hexane and benzene. The compound have a melting point of 247e252 C. The elemental analytical data of the antimicrobial compound produced by S. coeruleorubidus BTSS-301 showed the following C ¼ 66.91; H ¼ 8.42; N ¼ 5.57; O ¼ 19.10; this analysis indicates a suggested empirical formula of C14H21NO3.
3.7.
401
1464e1415 cm1 corresponds to CeC value in alcohol containing ring and the presence of band at 1384.37 cm1 corresponds to aromatic carboxylic acid. The absorption bands falling in the region 762.93e575.63 cm1 show the presence of aromatic hydrogen in the compound (Fig. 2). The 1H NMR spectrum was obtained at 399.7 MHz and 13C NMR spectra was obtained at 100.5 MHz. From the 1H NMR spectrum, chemical shifts were observed at 7.53e7.56 and 7.64e7.74, indicating the presence of a di substituted aromatic ring. The chemical shift value at 4.42 and 4.68 indicates the presence of eCHeOHeandeCHeNHegroups in the compound. The peaks at 1.24 to 1.80 corresponds to the presence of aliphatic hydrogens i.e, methyl and methylene groups. From the 13C NMR spectrum of the compound, peaks were observed at 10.93 and 14.02, which corresponds to methyl groups and peaks at 19.168, 22.638, 23.730, 28.904, 29.469, 30.344, 31.901, 34.379, and 38.709 represents the presence of different eCH2 groups. The peaks at 65.561 and 68.147 represents carbon atoms attached to a hetero atom i.e, CeO or CeN. The chemical shifts at 128.783 and 128.822, 130.866 and 132.431 correspond to the presence of aromatic ring system. Finally the peak at 167.756 belongs to C]O group of an amide or an ester. By the chemical assignments obtained from the spectral studies, the compound is not identical with similar antibiotics described in literature. The antimicrobial compound is therefore identified as N-ethyl-2-(2-(3-hydroxybutyl) phenoxy) acetamide and the probable structure is shown in (Fig. 3).
Spectroscopic characteristics 3.8.
The UV-visible spectra in methanol showed characteristics absorption spectra at l ¼ 207, 248 and 364. Among these strong UV absorption maxima was observed at 248 nm with a shoulder at 364 nm, thus suggesting a polyene nature of the compound. The infra-red spectrum showed absorption bands at 3421.45 cm1 may be due the presence of hydroxyl group in aromatic ring; bands at 2958e2851 cm1 are due the methyl or carboxylic stretch rings, respectively. Whereas, the band at 1730.99 cm1 is due the presence of C]O function of an ester or an amide group. Band at 1643.13 cm1 confirms the presence of C]C in 5 membered ring, bands at position
Table 3 e Effect of different amino acids on growth and metabolite production. Amino acids (2 mg/ml)
Growth in dry weight (mg/ml)
Antibiotic concentration (mg/ml)
Alanine Arginine Aspartate Asparagine Glutamate Leucine Histidine Methionine Phenylalanine Serine Threonine Tryptophan Tyrosine
2.0 2.6 2.3 2.5 2.6 1.6 2.2 1.8 2.0 2.3 2.0 2.6 2.3
128 170 142 156 160 110 144 120 135 138 122 148 136
Minimum inhibitory concentration
The purified compound showed broad spectrum of antimicrobial activity against selective Gram positive bacteria, Gram negative bacteria and fungi. The lowest MIC was recorded against E. coli and B. cereus (10 mg/ml) and highest against S. aureus (28 mg/ml). The MIC of fungi was lowest (35 mg/ml) for A. flavus and highest (86 mg/ml) for C. albicans (Table 4).
4.
Discussion
The results showed that, the growth and antimicrobial compound production was highest with glucose than that of other carbon sources used in the study. The maximum yield was obtained with 10 g/l concentration of glucose in the medium, while at 12.5 g/l glucose concentration the metabolite yield was relatively close to that of 10 g/l glucose concentration but the growth was less (3 mg/ml). Further, increase or decrease in glucose concentration reduced the growth and yield. Nitrogen source in addition to the carbon source also play an important role in the antibiotic production. In comparison with organic nitrogen sources, inorganic nitrogen sources produced more metabolite. The maximum yield was obtained with NH4NO3 at 2.5 g/l concentration in the medium, other nitrogen sources also favored good growth but the yield was less in comparison to NH4NO3. The results suggest that the level of antibiotic production may be greatly influenced by the nature and the type of the nitrogen source supplied in the culture medium. In addition to the carbon and nitrogen sources, addition of metal ions such as K2HPO4 at 2.0 g/l and MgSO4.7H2O at 1.0 g/l concentration strongly influenced the yield and enhanced the
402
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Fig. 2 e Infra-red absorption spectrum of the purified bioactive compound.
metabolite production. Further it is clear that above and below the critical concentrations of metal ions effect the growth and antibiotic production significantly. The isolate BTSS-301 showed a narrow range of incubation temperature for relatively good growth and production. The organism appeared to be mesophilic in nature with the optimum temperature of 30 C. The balanced use of carbon and nitrogen sources form the basis for pH control as buffering capacity is providing by the proteins, peptides and amino acids in the medium. The results evidently suggest that the isolate is capable of producing antimicrobial compound, only with in the optimum pH range (6.8e7.6) although; the strains withstands a broad range of pH (5.2e10.0).10 The results indicated that the optimum incubation period and agitation for maximum production was 96 h at 180 rpm. The yield was decreased at both lower and higher agitation speeds. The glucose uptake system of Streptomyces13 has revealed that the glucose permease gene could be found twice in genome, which specifies the uptake of glucose at higher concentrations than other carbon sources. Addition of ammonium as nitrogen source to the fermentation medium markedly increases the antibiotic production of AK-111-81 by S. hygroscopicus 111-81.14 Similarly it is used for the production of aureobasidins and antifungal antibiotic from T. harzianum15,16 respectively. James et al17 reported that the addition
of amino acids to the production medium acts as growth promoters and enhances antibiotic production. Several studies have revealed that the antimicrobial compound production was high at optimum concentrations of metal ions.18,19 However, an excessive amount of inorganic phosphate also suppressed the production of antibiotics such as, tetracycline, actinomycin, and candicidin.20 Present results also indicated the repression of bioactive compound production at higher phosphate concentration in the medium. Streptomyces usually produce antibiotics at temperature near 27 C. Generally the range of temperature supporting good growth is as wide as 25 C, but the temperature range adequate for good production of secondary metabolites is narrow i.e., 5e10 C.17 Spectroscopic analysis revealed that the compound has lmax at 207, 248 and 364. The IR spectral data revealed that the compound contains a carbonyl function of an ester or amide group, hydroxyl group, methyl stretch rings and aromatic hydrogen’s. The antimicrobial compound is therefore identified as N-ethyl-2-(2-(3-hydroxybutyl) phenoxy) acetamide. The MIC of the purified compound revealed its broad
Table 4 e Minimum inhibitory concentrations (MIC) of the purified bioactive metabolite isolated from Streptomyces coeruleorubidus BTSS-301. Test organisms
Fig. 3 e Probable structure of the purified antimicrobial compound has been identified as N-ethyl-2-(2-(3hydroxybutyl) phenoxy) acetamide.
Bacteria S. aureus (MTCC 3160) B. subtilis (MTCC 441) B. cereus (MTCC 430) P. aeruginosa (MTCC 424) E. coli (MTCC 443) P. vulgaris (MTCC 426) Fungi & yeast S. cerevisiae (MTCC 170) C. albicans (MTCC 227) A. niger (MTCC 961) A. flavus (MTCC 3396)
MIC (mg/ml) 28 mg/ml 18 mg/ml 10 mg/ml 24 mg/ml 10 mg/ml 20 mg/ml 50 86 42 35
mg/ml mg/ml mg/ml mg/ml
j o u r n a l o f p h a r m a c y r e s e a r c h 7 ( 2 0 1 3 ) 3 9 7 e4 0 3
spectrum of antimicrobial activity against Gram positive bacteria, Gram negative bacteria and fungi.
Conflicts of interest All authors have none to declare.
Acknowledgment The authors are grateful to Ministry of Earth Sciences, Government of India, New Delhi for financial assistance and thankful to Departments of Biochemistry, Organic chemistry, College of Science and Technology and College of Pharmaceutical Sciences, Andhra University for HPLC, IR and NMR studies. The authors are thankful to the JPR Solutions for providing partial funds in publishing this article.
references
1. Berdy J. Bioactive microbial metabolites. J Antibiot. 2005;58:1e26. 2. Fenical W, Jensen PR. Developing a new resource for drug discovery: marine actinomycete bacteria. Nat Chem Biol. 2006;2:666e673. 3. Stackebrandt E, Rainey FA, Ward-Rainey NL. Proposal for a new hierarchic classification system, Actinobacteria classis nov. Int J Syst Bacteriol. 1997;47:479e491. 4. Baltz RH. Renaissance in antibacterial discovery from actinomycetes. Curr Opin Pharmacol. 2008;8:557e563. 5. Theobald U, Schimana J, Fiedler HP. Microbial growth and production kinetics of Streptomyces antibioticus Tu 6040. Antonie Van Leeuwenhoek. 2000;78:307e313. 6. Omura S, Ikeda H, Ishikawa J, et al. Genome sequence of an industrial microorganism Streptomyces avermitilis: deducing the ability of producing secondary metabolites. Proc Natl Acad Sci U S A. 2001;98:12215e12220. 7. Goodfellow M, Fiedler HP. A guide to successful bioprospecting: informed by actinobacterial systematics. Antonie Van Leeuwenhoek. 2010;98:119e142.
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8. Shang K, Youjia H, Chunbao Z, Baoquan Z. Production of 40 epidaunorubicin by metabolic engineering of Streptomyces coeruleorubidus strain SIPI-1482. World J Microbiol Biotechnol. 2008;24:1107e1113. 9. Shirling EB, Gottlieb D. Methods for characterization of Streptomyces species. Int J Syst Bacteriol. 1966;16(3): 313e340. 10. Siva Kumar K, Haritha R, Swathi A, Sirisha B, Ramana T. Taxonomy and antimicrobial activity of Streptomyces coeruleorubidus sp. isolated from marine sediment. Res J Microbiol. 2012;7(3):171e181. 11. Pridham TG, Gottlieb D. The utilization of carbon compounds by some Actinomycetales as an aid for species determination. J Bacteriol. 1948;56:107e114. 12. Wiegand I, Hilpert K, Hancock REW. Agar and broth dilution methods to determine the minimal inhibitory concentration (MIC) of antimicrobial substances. Nat Protoc. 2008;3(2):163e175. 13. Van Wezel GP, Mahr K, Konig M, et al. GlcP constitutes the major glucose uptake system of Streptomyces coelicolor A3(2). Mol Microbiol. 2005;55:624e636. 14. Gesheva V, Ivanova V, Gesheva R. Effects of nutrients on the production of AK-111-81 macrolide antibiotic by Streptomyces hygroscopicus. Microbiol Res. 2005;160:243e248. 15. Takesako K, Ikai K, Haruna F, Endo M, Shimanaka K, Sone E. Aureobasidins, new antifungal antibiotics: taxonomy, fermentation, isolation and properties. J Antibiot. 1991;44:919e924. 16. Jayraj J, Ramabadran R. Effect of certain nitrogenous sources on the in vitro growth, sporulation and production of antifungal substances by Trichoderma hazianum. J Mycol Plant Pathol. 1998;28:23e25. 17. James PDA, Edwards C, Dawson M. The effects of temperature, pH and growth rate on secondary metabolism in Streptomyces thermoviolaceus grown in a chemostat. J Gen Microbiol. 1991;137:1715e1720. 18. Abbas AS, Edwards C. Effects of metals on Streptomyces coelicolor growth and actinorhodin production. Appl Environ Microbiol. 1990;56(3):675e680. 19. Young MD, Kempe LL, Badert FG. Effects of phosphate, glucose, and ammonium on cell growth and lincomycin production by Streptomyces lincolnensis in chemically defined Media. Biotechnol Bioeng. 1985;27:327e333. 20. Kishimoto K, Park YS, Okabe M, Akiyama S. Effect of phosphate ion on mildiomycin production by Streptoverticillium rimofaciens. J Antibiot. 1996;49:775e780.
Available online at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/jopr
Original Article
Production, purification and characterization of an antimicrobial compound from marine Streptomyces coeruleorubidus BTSS-301 Siva Kumar Kandula a,*, Ramana Terli b a
Research Scholar, Department of Biotechnology, College of Science and Technology, Andhra University, Visakhapatnam 530003, Andhra Pradesh, India b Dean of Life Sciences, GITAM Institute of Science, GITAM University, Visakhapatnam 530045, Andhra Pradesh, India
article info
abstract
Article history:
Background: An indigenous marine Streptomyces coeruleorubidus BTSS-301 has been isolated
Received 14 March 2013
from marine sediment sample near Visakhapatnam coast at a depth of 30 m. It exhibited
Accepted 27 April 2013
broad spectrum of antimicrobial activity. However, for maximum production of a specific
Available online 2 June 2013
bioactive compound from indigenous isolates medium formulation is an essential step. Methods: Optimization experiments for various carbon and nitrogen sources and cultural
Keywords:
parameters were performed by shake flask culture method. The active compound in cul-
Antimicrobial compound
ture filtrate was extracted into ethyl acetate and then purified by silica gel column chro-
Biomass
matography. The purified active compound was characterized by spectroscopy studies.
Medium optimization
The minimum inhibitory concentration was determined by broth dilution method.
Minimum inhibitory concentration
Results & discussion: Optimization studies revealed that the highest antibiotic production
Streptomyces coeruleorubidus
was obtained when glucose at 10.0 g/l and NH4NO3 at 2.5 g/l were used as carbon and nitrogen sources respectively. Cultural parameters revealed that 96 h of incubation at 30 C at 180 rpm with pH 7.2 of the production medium exhibited maximum yield. The purified active compound is identified as N-ethyl-2-(2-(3-hydroxybutyl) phenoxy) acetamide. Conclusion: The antimicrobial compound from S. coeruleorubidus BTSS-301 could be a candidate in the generation of new antimicrobial agents from marine Streptomyces. Copyright ª 2013, JPR Solutions; Published by Reed Elsevier India Pvt. Ltd. All rights reserved.
1.
Introduction
Streptomyces are the most economically and biotechnologically valuable prokaryotes. They are responsible for the production of about half of the discovered bioactive secondary metabolites such as antibiotics, antitumor agents and immunosuppressive agents.1 The identification of new
compounds from terrestrial Streptomyces has gradually decreased and the re-isolation of existing metabolites has increased.2 Thus, marine habitats were screened for novel bioactive secondary metabolites. As marine environmental conditions are extremely different from terrestrial ones, it is assumed that marine Streptomyces might produce different types of bioactive secondary metabolites.3,4 The success of
* Corresponding author. Tel.: þ91 9912286860; fax: þ91 (0) 891 2734821. E-mail address: [email protected] (S.K. Kandula). 0974-6943/$ e see front matter Copyright ª 2013, JPR Solutions; Published by Reed Elsevier India Pvt. Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jopr.2013.04.047
398
j o u r n a l o f p h a r m a c y r e s e a r c h 7 ( 2 0 1 3 ) 3 9 7 e4 0 3
screening programs for antibiotic production is heavily dependent on the identification of isolates to the correct taxa. However for indigenous isolates it is essential to grow in a diverse range of production media including the use of formulations which mimic conditions in the environment in the case of strains from marine habitats.5 Medium formulation is an essential stage for the successful production of a specific bioactive compound. The media used for submerged cultivation of Streptomyces have a dramatic impact on the expression of secondary metabolite gene clusters.6 The antibiotic production is highly based on the carbon/nitrogen ratio in the medium. Medium with a high content of both carbon and nitrogen source (3.5:1, C/N ratio) permits optimal growth of nearly all actinomycetes strains.7 Several clinically useful compounds were reported from Streptomyces coeruleorubidus. These include pacidamycins, antifungal metabolites, and indispensable anticancer drugs such as, doxorubicin and epirubicin.8 The present study was undertaken to examine the effect of different nutrients and cultural conditions on antimicrobial compound production and to purify extra cellular compound from the indigenous marine isolate S. coeruleorubidus BTSS-301 and to determine the structure of the purified compound.
2.
Methods
2.1.
Microorganism
The indigenous organism designated as BTSS-301, was isolated from a marine sediment sample collected from Bay of Bengal near Visakhapatnam coast at a depth of 30 m. Morphological, cultural and physiological characteristics of the strain were studied using the International Streptomyces Project (ISP) media recommended by Shirling and Gottlieb9 and was taxonomically characterized by using Polyphasic approach. The isolate has been identified as S. coeruleorubidus10 (Data published).
2.2.
2.4.
Basal medium
To determine the optimal nutritional and cultural conditions for growth and antimicrobial activity, Pridham and Gottlieb’s11 inorganic salt medium was used as the production medium base.
2.5. Optimization of media and cultural conditions for antimicrobial compound production The effect of various carbon sources, glucose concentration, organic nitrogen sources, inorganic nitrogen sources, NH4NO3 concentration, metal ions and cultural conditions were optimized by using shake flask culture method. The biomass from the culture filtrate was separated by means of centrifugation. It was transferred to pre weighed dry Whatman No. 1 filter paper. The filter paper along with the biomass was dried in a hot air oven at 80 C for 18e24 h to reach a fixed weight. Growth was expressed in terms of dry weight as mg/ml culture medium.
2.6.
Fermentation
The S. coeruleorubidus BTSS-301inoculum was introduced aseptically into sterile flasks containing ingredients (g/l) glucose, 10; NH4NO3, 2.5; K2HPO4, 2.0; MgSO4.7H2O, 1.0; and trace salt solutions9 1.0 ml, with pH of the medium 7.2. The flasks were incubated for 96 h at 30 C at 180 rpm. The culture filtrate was then separated by centrifugation at 3000 rpm for 15 min.
2.7. Extraction and purification by column chromatography
Test microorganisms
The following microorganisms procured from IMTECH, Chandigarh, India were used during the investigation as test microorganisms. Staphylococcus aureus (MTCC 3160), Bacillus subtilis (MTCC 441), Bacillus cereus (MTCC 430), Pseudomonas aeruginosa (MTCC 424), Escherichia coli (MTCC 443), Proteus vulgaris (MTCC 426), Saccharomyces cerevisiae (MTCC 170), Candida albicans (MTCC 227), Aspergillus niger (MTCC 961), and Aspergillus flavus (MTCC 3396).
2.3.
suspended in saline solution. 5 ml of this suspension was used as inoculum for the optimization experiments by shake flask culture.
Inoculum preparation
Seed medium composed of (g/l) soluble starch 25; Ammonium sulfate, 5; NaCl, 5; CaCO3, 5 with pH adjusted to 7.0 was used for the seed production. For the seed growth, mycelium from a seven day old, well-sporulated slant of the culture was inoculated into 200 ml of seed medium and grown at 28 C with 120 rpm on a shaker incubator for 48 h. Then culture was centrifuged at 3000 rpm for 10 min to separate the cells from the broth. The cell pellet was washed thoroughly and
The culture filtrate (12 l) was extracted twice with ethyl acetate and the pooled solvent extracts were evaporated to dryness under reduced pressure to yield a crude residue. The purification of the antimicrobial compound was carried by using silica gel column (2.5 25) chromatography. Silica gel of 100e200 mm particle size was used for packing the column. Chloroform and methanol (7:3, v/v) were used as an eluting solvent. 5 g of crude extract to be fractioned was dissolved in 50 ml of methanol and passed through the silica gel column keeping the flow rate at 0.2 ml/min; thirty fractions were collected (5 ml each) and tested for their antimicrobial activities. The purity of the active fraction was determined by Waters Reverse Phase HPLC, Spherisorb 5 mm ODS 2 (C18) column with solvent system methanol and water 70:30 (v/v) at 2500 psi in isocratic mode. The operating flow rate was 1.0 ml/min. The solubility pattern of the compound was determined in various polar and non-polar solvents. The melting point of the compound was determined by FishereJohns melting point apparatus.
j o u r n a l o f p h a r m a c y r e s e a r c h 7 ( 2 0 1 3 ) 3 9 7 e4 0 3
2.8.
Spectroscopic analysis
The UV absorption spectrum of the compound was determined by Shimadzu UV 1800 spectrophotometer. The Infrared (IR) spectrum of the purified antimicrobial compound was recorded using Bruker Alpha FT-IR spectroscopy. The resulting data generated was viewed with the help of OPUS v6.5 software. NMR spectrum of the compound was determined by using an AMX-400 spectrometer (Bruker, Germany) 1 H data was obtained at 399.7 MHz and 13C was at 100.5 MHz using chloroform-d as solvent and trimethylsilane as internal reference.
2.9.
Minimum inhibitory concentration (MIC)
The minimum inhibitory concentration has been determined by broth dilution method.12 The media used were nutrient broth for bacteria and Czapek Dox broth for fungi.
3.
Results
3.1.
Effect of carbon source
3.2.
Effect of nitrogen source
Among the various inorganic nitrogen sources, the maximum metabolite production was achieved with
Table 1 e Effect of different carbon sources on growth and metabolite production. Carbon sources 1% (w/v) D-Glucose D-Fructose D-Galactose D-Xylose Arabinose Glycerol Starch Maltose Lactose Sucrose Meso-inositol Mannitol
NH4NO3 (192 mg/ml) with biomass of 3.8 mg/ml. Among the organic nitrogen sources, the high level of metabolite yield was obtained with soyabean meal (Table 2). Further, the concentration of 2.5 g/l of NH4NO3 (Fig. 1) greatly influenced the antimicrobial compound production with maximum yield and biomass accretion of 3.3 mg/ml. Moreover the yield was reduced with increase and decrease of NH4NO3 concentration.
3.3.
Growth in dry weight (mg/ml)
Antibiotic concentration (mg/ml)
3.0 0.8 0.8 0.4 0.5 2.4 2.2 0.8 0.7 0.8 0.6 0.3
160 50 58 38 44 112 120 20 18 50 48 35
Effect of amino acids
Different amino acids were added to the improved production medium containing glucose and NH4NO3 as carbon and nitrogen sources. Out of 13 amino acids, only arginine, glutamate, asparagine, aspartate, tryptophan and histidine favored the growth and metabolite production (Table 3). Among them, arginine, glutamate and tryptophan promoted the maximum biomass accumulation (2.6 mg/ml) than the other amino acids. The remaining amino acids yielded relatively less amount of antibiotic.
3.4.
The optimization of the metabolite production was carried out in batch cultures. The isolate BTSS-301 was cultivated in basal medium supplemented with different carbon sources, and their effect on growth and antimicrobial activity was studied (Table 1). The isolate grow in all the test carbon sources. Maximum metabolite production was obtained with glucose (160 mg/ml) followed by glycerol (120 mg/ml) and starch (112 mg/ ml) and the biomass obtained was also highest with glucose (3 mg/ml) than that of glycerol and starch. The effect of different concentrations of glucose (Fig. 1) on growth and production showed that the antibiotic titer was highest with 10 g/l glucose concentration with biomass of 3.6 mg/ml.
399
Effect of metal ions
The maximum biomass (3.6 mg/ml) and metabolite production was favored at 2.0 g/l concentration of K2HPO4 (Fig. 1). Similarly the effect of different concentrations of MgSO4.7H2O on growth and metabolite yield was also studied. The results indicate that the concentration of both the metal ions strongly influence the antibiotic production. The concentration of 1.0 g/l MgSO4.7H2O promoted the maximum growth (3.2 mg/ ml) and antimicrobial compound production (Fig. 1).
3.5.
Effect of cultural parameters
In addition to culture media, cultural conditions strongly influence the antimicrobial compound production. The effect of cultural conditions on growth and production by the isolate BTSS-301 has been studied in detail. Maximum antibiotic yield was obtained at 30 C with biomass of 3.6 mg/ml (Fig. 1). The increase of incubation temperature from 20 C to 30 C increased the growth of biomass and the production of metabolite respectively. However, the yield decreased consistently with the cell mass by increasing the growth temperature range from 35 to 50 C. Even though biomass was deposited at 45e50 C, the antibiotic yield was negligible. The maximum antibiotic yield was obtained at pH 7.2 with a biomass of 2.8 mg/ml (Fig. 1). The growth and antibiotic production by the isolate BTSS-301 was monitored over a period of 120 h. The antibiotic production occurred in a growth phase dependent manner and the highest yield was obtained in the late exponential phase and the stationery phase. The maximum yield was obtained at 96 h incubation period with biomass of 3.9 mg/ml (Fig. 1). The agitation provides greater aeration to the culture and also creates conditions for greater availability of nutrients to cells. The highest metabolite yield was obtained at 180 rpm with biomass of 3.2 mg/ml (Fig. 1). Further increase in the agitation speed demonstrated rapid decrease in yield along with biomass.
400
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Fig. 1 e Effect of nutrients, metal ions and cultural conditions on biomass and metabolite production.
3.6.
Fermentation, extraction and purification
The fermentation process was carried out for 96 h at 30 C. After incubation period, the culture supernatant was separated by centrifugation at 3000 rpm for 15 min. the culture filtrate was extracted twice with ethyl acetate (1:2, v/v) and the organic layer was evaporated to dryness under reduced pressure to give yellow colored precipitate. 5 g of the precipitate in 50 ml of methanol was chromatographed on silica gel column using solvent system chloroform and methanol (7:3, v/v). A total of 30 fractions of 5 ml each were collected. Among all the fractions tested for antimicrobial activity, active fractions were ranged between fraction no.11e23. Further
purification was carried out by silica gel column chromatography to remove minor impurities and unsolicited metabolites. A total of 20 minor fractions of 2 ml each were collected. All of them were subjected to TLC analysis and fractions with similar Rf (0.69) values were pooled together. Finally three major fractions were obtained IIIa (232 mg), IIIb (23 mg) and IIIc (10 mg). Out of these three fractions, fraction IIIa exhibited highest antimicrobial activity when compared with the remaining two fractions. The purity of the active fraction was analyzed by reverse phase HPLC, confirming the 95% purity of the compound. The compound was obtained in form of a crystalline yellow colored solid material. It was soluble in DMSO, methanol,
j o u r n a l o f p h a r m a c y r e s e a r c h 7 ( 2 0 1 3 ) 3 9 7 e4 0 3
Table 2 e Effect of different nitrogen sources on growth and metabolite production. Nitrogen sources
Growth in dry weight (mg/ml)
Inorganic nitrogen sources (0.2% w/v) Ammonium citrate 1.6 Ammonium nitrate 3.8 Ammonium sulfate 1.9 Potassium nitrate 1.5 Sodium nitrate 1.8 Organic nitrogen sources (0.2% w/v) Soyabean meal 3.1 Peptone 1.5 Beef extract 1.9 Yeast extract 2.4 Tryptone 1.8
Antibiotic concentration (mg/ml) 122 192 155 98 138 192 89 142 160 110
ethanol, acetone, ethyl acetate, chloroform, and diethyl ether but insoluble in hexane and benzene. The compound have a melting point of 247e252 C. The elemental analytical data of the antimicrobial compound produced by S. coeruleorubidus BTSS-301 showed the following C ¼ 66.91; H ¼ 8.42; N ¼ 5.57; O ¼ 19.10; this analysis indicates a suggested empirical formula of C14H21NO3.
3.7.
401
1464e1415 cm1 corresponds to CeC value in alcohol containing ring and the presence of band at 1384.37 cm1 corresponds to aromatic carboxylic acid. The absorption bands falling in the region 762.93e575.63 cm1 show the presence of aromatic hydrogen in the compound (Fig. 2). The 1H NMR spectrum was obtained at 399.7 MHz and 13C NMR spectra was obtained at 100.5 MHz. From the 1H NMR spectrum, chemical shifts were observed at 7.53e7.56 and 7.64e7.74, indicating the presence of a di substituted aromatic ring. The chemical shift value at 4.42 and 4.68 indicates the presence of eCHeOHeandeCHeNHegroups in the compound. The peaks at 1.24 to 1.80 corresponds to the presence of aliphatic hydrogens i.e, methyl and methylene groups. From the 13C NMR spectrum of the compound, peaks were observed at 10.93 and 14.02, which corresponds to methyl groups and peaks at 19.168, 22.638, 23.730, 28.904, 29.469, 30.344, 31.901, 34.379, and 38.709 represents the presence of different eCH2 groups. The peaks at 65.561 and 68.147 represents carbon atoms attached to a hetero atom i.e, CeO or CeN. The chemical shifts at 128.783 and 128.822, 130.866 and 132.431 correspond to the presence of aromatic ring system. Finally the peak at 167.756 belongs to C]O group of an amide or an ester. By the chemical assignments obtained from the spectral studies, the compound is not identical with similar antibiotics described in literature. The antimicrobial compound is therefore identified as N-ethyl-2-(2-(3-hydroxybutyl) phenoxy) acetamide and the probable structure is shown in (Fig. 3).
Spectroscopic characteristics 3.8.
The UV-visible spectra in methanol showed characteristics absorption spectra at l ¼ 207, 248 and 364. Among these strong UV absorption maxima was observed at 248 nm with a shoulder at 364 nm, thus suggesting a polyene nature of the compound. The infra-red spectrum showed absorption bands at 3421.45 cm1 may be due the presence of hydroxyl group in aromatic ring; bands at 2958e2851 cm1 are due the methyl or carboxylic stretch rings, respectively. Whereas, the band at 1730.99 cm1 is due the presence of C]O function of an ester or an amide group. Band at 1643.13 cm1 confirms the presence of C]C in 5 membered ring, bands at position
Table 3 e Effect of different amino acids on growth and metabolite production. Amino acids (2 mg/ml)
Growth in dry weight (mg/ml)
Antibiotic concentration (mg/ml)
Alanine Arginine Aspartate Asparagine Glutamate Leucine Histidine Methionine Phenylalanine Serine Threonine Tryptophan Tyrosine
2.0 2.6 2.3 2.5 2.6 1.6 2.2 1.8 2.0 2.3 2.0 2.6 2.3
128 170 142 156 160 110 144 120 135 138 122 148 136
Minimum inhibitory concentration
The purified compound showed broad spectrum of antimicrobial activity against selective Gram positive bacteria, Gram negative bacteria and fungi. The lowest MIC was recorded against E. coli and B. cereus (10 mg/ml) and highest against S. aureus (28 mg/ml). The MIC of fungi was lowest (35 mg/ml) for A. flavus and highest (86 mg/ml) for C. albicans (Table 4).
4.
Discussion
The results showed that, the growth and antimicrobial compound production was highest with glucose than that of other carbon sources used in the study. The maximum yield was obtained with 10 g/l concentration of glucose in the medium, while at 12.5 g/l glucose concentration the metabolite yield was relatively close to that of 10 g/l glucose concentration but the growth was less (3 mg/ml). Further, increase or decrease in glucose concentration reduced the growth and yield. Nitrogen source in addition to the carbon source also play an important role in the antibiotic production. In comparison with organic nitrogen sources, inorganic nitrogen sources produced more metabolite. The maximum yield was obtained with NH4NO3 at 2.5 g/l concentration in the medium, other nitrogen sources also favored good growth but the yield was less in comparison to NH4NO3. The results suggest that the level of antibiotic production may be greatly influenced by the nature and the type of the nitrogen source supplied in the culture medium. In addition to the carbon and nitrogen sources, addition of metal ions such as K2HPO4 at 2.0 g/l and MgSO4.7H2O at 1.0 g/l concentration strongly influenced the yield and enhanced the
402
j o u r n a l o f p h a r m a c y r e s e a r c h 7 ( 2 0 1 3 ) 3 9 7 e4 0 3
Fig. 2 e Infra-red absorption spectrum of the purified bioactive compound.
metabolite production. Further it is clear that above and below the critical concentrations of metal ions effect the growth and antibiotic production significantly. The isolate BTSS-301 showed a narrow range of incubation temperature for relatively good growth and production. The organism appeared to be mesophilic in nature with the optimum temperature of 30 C. The balanced use of carbon and nitrogen sources form the basis for pH control as buffering capacity is providing by the proteins, peptides and amino acids in the medium. The results evidently suggest that the isolate is capable of producing antimicrobial compound, only with in the optimum pH range (6.8e7.6) although; the strains withstands a broad range of pH (5.2e10.0).10 The results indicated that the optimum incubation period and agitation for maximum production was 96 h at 180 rpm. The yield was decreased at both lower and higher agitation speeds. The glucose uptake system of Streptomyces13 has revealed that the glucose permease gene could be found twice in genome, which specifies the uptake of glucose at higher concentrations than other carbon sources. Addition of ammonium as nitrogen source to the fermentation medium markedly increases the antibiotic production of AK-111-81 by S. hygroscopicus 111-81.14 Similarly it is used for the production of aureobasidins and antifungal antibiotic from T. harzianum15,16 respectively. James et al17 reported that the addition
of amino acids to the production medium acts as growth promoters and enhances antibiotic production. Several studies have revealed that the antimicrobial compound production was high at optimum concentrations of metal ions.18,19 However, an excessive amount of inorganic phosphate also suppressed the production of antibiotics such as, tetracycline, actinomycin, and candicidin.20 Present results also indicated the repression of bioactive compound production at higher phosphate concentration in the medium. Streptomyces usually produce antibiotics at temperature near 27 C. Generally the range of temperature supporting good growth is as wide as 25 C, but the temperature range adequate for good production of secondary metabolites is narrow i.e., 5e10 C.17 Spectroscopic analysis revealed that the compound has lmax at 207, 248 and 364. The IR spectral data revealed that the compound contains a carbonyl function of an ester or amide group, hydroxyl group, methyl stretch rings and aromatic hydrogen’s. The antimicrobial compound is therefore identified as N-ethyl-2-(2-(3-hydroxybutyl) phenoxy) acetamide. The MIC of the purified compound revealed its broad
Table 4 e Minimum inhibitory concentrations (MIC) of the purified bioactive metabolite isolated from Streptomyces coeruleorubidus BTSS-301. Test organisms
Fig. 3 e Probable structure of the purified antimicrobial compound has been identified as N-ethyl-2-(2-(3hydroxybutyl) phenoxy) acetamide.
Bacteria S. aureus (MTCC 3160) B. subtilis (MTCC 441) B. cereus (MTCC 430) P. aeruginosa (MTCC 424) E. coli (MTCC 443) P. vulgaris (MTCC 426) Fungi & yeast S. cerevisiae (MTCC 170) C. albicans (MTCC 227) A. niger (MTCC 961) A. flavus (MTCC 3396)
MIC (mg/ml) 28 mg/ml 18 mg/ml 10 mg/ml 24 mg/ml 10 mg/ml 20 mg/ml 50 86 42 35
mg/ml mg/ml mg/ml mg/ml
j o u r n a l o f p h a r m a c y r e s e a r c h 7 ( 2 0 1 3 ) 3 9 7 e4 0 3
spectrum of antimicrobial activity against Gram positive bacteria, Gram negative bacteria and fungi.
Conflicts of interest All authors have none to declare.
Acknowledgment The authors are grateful to Ministry of Earth Sciences, Government of India, New Delhi for financial assistance and thankful to Departments of Biochemistry, Organic chemistry, College of Science and Technology and College of Pharmaceutical Sciences, Andhra University for HPLC, IR and NMR studies. The authors are thankful to the JPR Solutions for providing partial funds in publishing this article.
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