R.Sugumaran et al.

, IJSID, 2012, 2 (6), 551- 558

ISSN:2249-5347

IJSID

International Journal of Science Innovations and Discoveries

Research Article
1 Department

An International peer Review Journal for Science

Available online through www.ijsidonline.info

STUDIES ON THE BIO-ACTIVE COMPOUND FROM STREPTOMYCES FLAVOFUSCUS of Microbiology, PSG College of Arts and Science, Coimbatore, Tamil Nadu, India R. Sugumaran * D. Chithradevi P. Muthukrishnan

Received: 26-11-2012 Accepted: 24-12-2012

*Corresponding Author

ABSTRACT farming soil, to determine the antibacterial activity against different human pathogens. Totally, 9 psychrophilic actinomycetes were isolated and 5 were found to be facultative A recent study was carried out to isolate the psychrophilic actinomycetes from

psychrophiles and other 4 were mesophilic. The isolated psychrophilic strains were selected to determine antimicrobial activity against human pathogens. Actinomycetes marscescens, Acinetobacter sp., Streptococcus mutants, Bacillus cereus, Shigella flexineri, Vibrio cholerae, Salmonella typhi, Klebsiella pneumoniae, Pseudomonas auerogenosa, Proteus activity towards INTRODUCTIONBioactive compounds were separated by thin layer the test pathogen. chromatography and identified using Gas

isolates were identified as (Micromonospora sp., Streptomycetes sp., Nocadia sp,). The active culture filtrate was tested against Escherichia coli, Stapyhlococcus aureus, Serratia Address: Name: R. Sugumaran Place: Tamilnadu, India E-mail: sugu.raffish@gmail.com Micromonospora sp. (FS1 & FS2) and Streptomyces sp. (FS5) able to show more antibacterial vulgaris. All the actinomycetes isolates were able to produce secondary metabolites but analysis (GC-MS). Various compounds were analysed by GC-MS, from that the bioactive compound Stigmasta-5, 22-dien-3-ol, (3a, 22E) - (CAS) were found to be an active substance Chromatograpy - Mass Spectrophotometry

identified from Onyza floribunda and thus active against Streptococcus pneumoniae,

INTRODUCTION Staphylococcus aureus and Escherischia coli.

compound

Keywords: Streptomyces flavofuscus, antibacterial, human pathogens, GC-MS, Bioactive

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R.Sugumaran et al., IJSID, 2012, 2 (6), 551- 558 bioactive metabolites [1]. Among actinomycetes, the member of the genus Streptomyces are considered economically important because they alone constitute 50% of soil actinomycetes are produced by this genus [2]. They continue to be prolific or other pharmaceutically useful compounds [3]. Streptomyces spp are soil-dwelling filamentous bacteria with acomplex cycle of morphological differentiation [4]. Members of the genus Streptomyces are potential sources for secondary metabolites possessing a variety of biological activities, including antibacterial activity, which is used for human and animal treatment. It is estimated this bacteria synthesizes more than 7,000 metabolites [5]. Majority of Streptomycetes and other actinomycetes peptides, polyenes and tetracyclines. members produce a diverse array of antibiotics including aminoglycosides, glycopeptides, B-lactams, macrolides, nucleosides, a virulent pathogen responsible for a wide range of infections including pimples, pneumonia, osteomyelitis, endocarditis and bacteremia, has developed resistance to most classes of antibiotics; methicillin and vancomycin [8]. Rising numbers of the available antibiotics, so there is in need of screening actinomycetes for antimicrobial compounds [10]. against multiple drug resistant bacteria, was selected for biochemical and molecular characterization. MATERIALS AND METHODS Sample collection: a sterile glass container and stored at room temperature (30 oC). Clinical samples: resistant microbes towards antibiotic has cause for the unresponsive infectious disease in human being [9]. Emerging of novel Bioactive compounds like macrocyclic lactum, macrolide, quinine and peptide are present in actinomycetes. These Actinomycetes are gram-positive bacteria that are wide spread in nature and play a pivotal role in the production of INTRODUCTION

sources of secondary metabolites with biological activities that ultimately find application as antimicrobial, anticancer agents

Antibiotic resistant bacteria emerge clinically within months to years following their use [6, 7]. Staphylococcus aureus,

antibiotic classes has the strategy to control the emerging drug-resistant pathogen. Microbes gaining more resistant towards Streptomyces spp from soil and tested for antibacterial activity against bacterial sample. Strain FS4, showing potent activity

compounds have range of activity like antibacterial, antifungal, antitumor etc [11, 12 & 13]. This study aimed to isolate

Soil samples were collected from different agricultural places in and around south zone of Tamilnadu (Coimbatore) in

antibacterial activity were carried against the test organisms. The selective human pathogenic organisms used for antagonistic Shigella flexineri, Vibrio chlorae, Salmonella typhi, Klebsiella pneumoniae, Pseudomonas aurogenosa, Proteus vulgaris. Isolation of Actinomycetes from soil sample:

study were Escherichia coli, Stapyhlococcus aureus, Serratia marscescens, Acinetobacter sp., Streptococcus sp., Bacillus subtillis,

The test organisms were collected from PSG Institute of Medical Research and Science, Coimbatore and the

taken and inoculated in nutrient agar by spread plate method and the plates were incubated at 30 oC for 3 days until the colonies were developed. Based on the colony nature, the actinomycetes were isolated and inoculated into actinomycetes isolation agar. The plates were incubated at 30oC for a period of 15 days until the growth was observed. The grown colonies were purified by sub culturing on starch casein nitrate agar supplemented with cyclohexamide (0.01 mg/ml). International Journal of Science Innovations and Discoveries, Volume 2, Issue 6, November-December 2012

One gram of soil sample was serially diluted up to 10 -8 dilution. The dilutions such as 10-3, 10-4, 10-5 and 10-6 were

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R.Sugumaran et al., IJSID, 2012, 2 (6), 551- 558 Biochemical characteristics: Indole, Methyl red, Vogus-proskauer, Citrate utilization, Starch hydrolysis, Catalase and Oxidase tests Sugar utilization tests mannitol, xylose and sucrose [14]. Mass production: Various biochemical tests were carried out to characterize the type of actinomycetes and to identify the potent strain;

were performed to determine the ability of the organism to utilize various sugars as a carbon source such as glucose, lactose, production media (ISP-4) consist of soluble starch 10gm, Dipotassium phosphate 1gm, Magnesium phosphate 1gm, Sodium chloride 1gm, Calcium carbonate 3gm, Ferrous sulphate 1gm, Manganese chloride 1gm, Zinc sulphate 1gm in 1000 ml of 7.4. Biological activity: The actinomycetes were inoculated into the ISP-4 medium and kept in shaker for 15 days at 200-250 rpm. The

distilled water with 7.4 pH. The growth of organism was observed at optimal temperature 27-30oC and the optimum pH were free supernatant was collected and filtered with whatmann no.1 filter paper. Equal volume of ethyl acetate was added to supernatant, shaked well for 20 minutes and allowed to settle overnight. The aqueous layer was collected in watch glass and muller hinton agar and kept undisturbed for 20 minutes. The extracted bioactive compound mixture was added to each well at zones were measured. kept in hot air oven at 40oC for 4 hours. The dried compound was scrapped, mixed with distilled water and antibacterial activity was seen against human pathogen by agar well diffusion method. The test pathogenic organisms were swabbed in After 15 days, the culture was taken and centrifuged at 3000-5000 rpm for 10 minutes. After centrifugation, the cell

different concentration (50 l, 100 l & 200 l). The plates were incubated at 37 oC for 24 hours and the diameters of inhibition

International Journal of Science Innovations and Discoveries, Volume 2, Issue 6, November-December 2012

Figure 1: Scheme for the extraction of bioactive compound Fermentation Broth (50 ml)

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R.Sugumaran et al., IJSID, 2012, 2 (6), 551- 558 The active compound was separated on silica G 60 grade absorbent by mixture of chloroform and methanol (4:1)

Extraction, Purification and identification of bioactive compound from the strain:

solvent. The dried cellular filtrate was mixed with DMSO (Dimethyl sulfoxide) and 10 l (30mg/ml) of mixture was loaded in to iodine vapour for the identification of compounds as spot and the R f value was calculated [15]. MS system was equipped with a fused silica capillary column (DB 5 - MS Capillary Standard Non - Polar Column) was used to analyse the compound. The data was processed with GC/MS DSQ II (Thermo GC-MS DSQ II). Column condition was chromatography were subjected to mass-spectral analysis. The spectra were analyzed from the available library data, Trace Ultra search (version 5.0). sample and identified as Micromonospora sp., Streptomycetes sp., and Nocardiopsis sp., based on their morphological nature on the Actinomycetes isolation agar and SCNA plates and it revealed the most of the strains belonging to the white and grey colour and rough nature (Table: 1). positive reaction to MR and showing the ability to hydrolyze the urea but showing negative reaction for Indole and VP test FS4 and FS5 showed marked antibacterial activity against selected bacterial pathogens (Table: 4). crude extract of FS4 was analysed by TLC and pure single district band was exhibited. Strain code FS1 FS2 FS3 FS4 FS5 Morphology of hyphae Separate hyphae Rarely branched, separate hyphae Spiral, twisted hyphae Long hyphae, branched Long chain spore Mycelium colour Pink Grey to white Sandal Grey White Type of spore Monospore Monospore Long spore Various biochemical tests were proceeded to characterize the isolated strains. Most of the strains were showed The samples were collected from farming land around Coimbatore area. Totally 5 strains were isolated from the soil RESULT AND DISCUSSION

TLC plate. The TLC plate was kept in solvent until the solvent run of the plate. The TLC plates were UV radiated and exposed

programmed as column oven temperature 70oC rose to 260o C at 6oC /Min [16]. The peaks of compounds in gas

The partially purified active compound was analysed by gas liquid chromatography-mass spectrometry (GC-MS). GC-

(Table: 2). Strains FS1 to FS9 utilize glucose, sucrose and xylose as their carbon source (Table: 3). Among the 5 isolates FS1, crude extract was obtained from 50 ml of fermentation broth after defatted with ketoconozole. Totally 5 strain were isolated Table 1: Study of phenotypic characterization of isolated actinomycetes Small spore Long spore

and one strain found to effective towards the test organisms. The effective strain was taken and further studies were done. The Micromonospora sp., Micromonospora sp., Streptomyces sp., Norcadia sp., Streptomyces sp., Possible genera Gram positive + + + + +

The scheme for the extraction of bioactive compounds from the stain is presented in Figure1. An amount of 0.4 g

Stigmasta-5, 22-dien-3-ol, (3a, 22E) - (CAS). The probability of the compound was 60.50 and the molecular weight was 412. pneumoniae, Staphylococcus aureus and Escherischia coli (Manguro et al., 2010). International Journal of Science Innovations and Discoveries, Volume 2, Issue 6, November-December 2012

This compound was identified from plant onyza Floribunda a resource says this compound has activity against Streptococcus

The compound were analysed by GC-MS (Fig. 2). The peak obtained at 45.22 shows the presence of compound

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culture of the stain and biological activities were studied against gram-positive and gram-negative bacteria. The partially purified bioactive compound was analysed by GC-MS. Stigmasta-5, 22-dien-3-ol, (3a, 22E) - (CAS) showed strong antibacterial from actinomycetes. Strain code FS1 FS2 FS3 FS4 FS5 Table 2: Biochemical properties of isolated actinomycetes Indole MR + + + VP + Citrate + + + activity towards test organisms. The Stigmasta-5, 22-dien-3-ol, (3a, 22E) - (CAS) produced by this are not reported earlier AFB Catalase + + + + + Mannitol + ++ + ++ ++ Oxidase + + + -

In the present study, the bioactive compound Stigmasta-5, 22-dien-3-ol, (3a, 22E) - (CAS) were purified from the broth

R.Sugumaran et al., IJSID, 2012, 2 (6), 551- 558

Strain code FS1 FS2 FS3 FS4 FS5

Table: 3 Utilization of various carbohydrates by isolated actinomycetes Glucose ++ +++ ++ +++ ++ Lactose +++ + ++ + +

Key: + positive, - negative

Table 4: Antagonistic effect of actinomycetes against human pathogens

Key: +++ Strong, ++ moderate, + weak

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R.Sugumaran et al., IJSID, 2012, 2 (6), 551- 558

Figure 1: Scanning electron microscopic image of Streptomyces flavofuscus

Figure 2: Dried secondary metabolites International Journal of Science Innovations and Discoveries, Volume 2, Issue 6, November-December 2012

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R.Sugumaran et al., IJSID, 2012, 2 (6), 551- 558

Figure 4: Iodine exposed TLC plate with bioactive compound

Figure 5: GC-Spectra of an Active fraction: Presence of an Active compound Stigmasta-5, 22-dien-3-ol, (3a, 22E) - (CAS) at the International Journal of Science Innovations and Discoveries, Volume 2, Issue 6, November-December 2012 retention time 45.22.

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R.Sugumaran et al., IJSID, 2012, 2 (6), 551- 558 compound can be used as antibiotic in pharmacological industries. Stigmasterol is one of the groups of plant hormone that include -sitosterol, campesterol and ergosterol (provitamin D 2). Stigmasterol is an unsaturated sterol occurs in plant fats and have indicated that stigmasterol may be useful in prevention of cancer like ovarian, prostate, breast and colon cancers. ACKNOWLEDGEMENT providing necessary facilities for the completion of this work. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. The authors would like to thank the Management, Secretary and the Principal of PSG College of Arts and Science for REFERENCES in number of medicinal herbs. Stigamasterol is used as a precursor in manufacturing semisynthetic progesterone. Researchers Bioactive compound produced from Streptomyces flavofuscus were active against the test organism. The active CONCLUSION

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