Extraction, separation and characterization of bioactive compounds produced by Streptomyces isolated from vermicast soil
R. Balachandar1*, N. Karmegam2, R. Subbaiya3
1Research Scholar, Faculty of Bioengineering, Sathyabama Institute of Science and Technology, Rajiv Gandhi Salai, Chennai-600 119, Tamil Nadu, India.
2Department of Botany, Government Arts College, Salem-636 007, Tamil Nadu, India.
3Associate Professor, Department of Biotechnology, Vinayaka Mission’s Research Foundation, Aarupadi Veedu Institute of Technology, Paiyanoor, Chennai- 600 128, Tamil Nadu, India.
*Corresponding Author E-mail:
ABSTRACT:
This present study was aimed to investigate identification of potent actinomycetes that have the ability to produce bioactive compounds was isolated from the vermicast soil collected from Sirumali Hills South India. The ethyl acetate extraction of isolated Streptomyces showed high biological activity against human pathogens. The crude ethyl acetate extract was subjected to TLC to separate compounds. GC-MS and FTIR analaysis were carried out to reveal the structural elucidation of isolated compounds. The identification of bioactive compounds is based on the peak area and retention time. GC-MS chromatogram of extract showed the presence of six bioactive compounds, including saturated and unsaturated fatty acids. The antagonistic activity of Streptomyces sample might be due to the synergistic effect of the identified compounds.
KEYWORDS: Actionmycetes, vermicast, GC-MS, Bioactive compounds, Ethyl acetate extract.
INTRODUCTION:
Nowadays a steady increase in antibiotic resistance during past several decades, available antimicrobial agents are not sufficient to control the microbial infections. Drug resistance of microorganism was developed gradually due to the improper handling of clinical pathogens1,2, potential environmental contamination from chemical fungicides. There are more than 200 known diseases transmitted by bacteria, fungi, viruses and prions, rickettsia and other microbes to humans3,4. Therefore, it is good to turn an eye to nature to find antagonistic microorganisms and metabolites5. In the world over two third of useful antibiotics are natural origins and are produced by actinomycetes6, among this Streptomyces are major producers7. They are widely distributed and are next to bacteria in the order of abundance in soil8.
Many research works have been carried out to identify new antimicrobial agents9, 10, 11. Soil microbes are the most important natural sources exhibiting strong biological activity against a wide range of pathogens12. In general microbes produce bioactive molecules which are not supporting for their growth but useful to defend other microbes13. Soil microorganisms in particular are intensively exploited14.
Actionmycetes are belonging to Gram +ve microorganisms, filamentous with 60 % GC content in its genome15,16,17,18. They have the ability to produce wide variety of secondary metabolites, which are effective against human pathogens. Streptomyces are well known producers of various potential secondary metabolites, which have various biological activities such as antifungal, antibacterial, enzymatic, anti parasitic, antitumor and immunosuppressive actions19. Streptomyces are called as antibiotic store room. They also play a crucial role in protecting plants against phytopathogens20. Thousands of antibiotics were isolated from Streptomyces, these represents only a small fraction of bioactive compounds produced21,22. Therefore identification of Streptomyces from natural resources and characterization of bioactive compounds is valuable endeavour. This study was aimed at the isolation and extraction of potent bioactive compounds from Streptomyces species. Furthermore, the bioactive compound was characterized by GC-MS and FT-IR analysis.
MATERIALS AND METHODS:
Vermicast soil samples were collected from different places, kept in sterile plastic bags and transported to laboratory23. These soil samples were air dried and crushed for isolation following Sadauon et al. method24. The actinomycetes were isolated by standard serial dilution method. One gram of soil was dissolved in 9ml sterile water containing boiling tube. The dilution was carried upto 10-5 dilutions. After dilution 0.1ml of aliquot were spread on Actinomycetes Isolation Agar containing nalidixic acid (100mg/l) and ketoconzole (30mg/l) and incubated at 30ºC for 7 to 10 days25,26. Based on the morphological character the actinomycetes were isolated and further purified by subculture on ISP2 (International Streptomyces project medium 2.
Morphological identification of actinomycetes:
Cultural and morphological character of isolates were characterized following the directions given by ISP2 and bergy’s manual systamatic bacteriology27. The shape of the cell morphological character were observed under various media such as Actionmycetes isolation agar, Strach casein agar, Streptomyces agar etc. After incubation at 30ºC for 7-10 days the results were recorded.
Preliminary screening for antimicrobial activity:
The isolated actinomycetes samples were subjected to screening the ability of antimicrobial activity by performing cross streak method28. Isolated samples were inoculated at the centre of AIA plate and incubated at 30ºC for 7-10 days. Then the plates were inoculated with test human pathogens by single streak around the actinomycetes sample and incubated at 37ºC overnight. After incubation the best isolate which showed a good antimicrobial activity was selected29.
Inoculum preparation:
Potent antimicrobial compound producing actionomycetes screened from preliminary screening were subjected to scale up inoculums preparation. Erlenmeyer flasks containing starch casein nitrate broth, pH-7 was inoculated with small disks taken from 7day old culture30. The inoculated flasks were incubated at 30ºC for 48-72 hrs in a rotary shaker. 50ul of inoculum was inoculated in 500 conical flasks with previously prepared medium. The inoculated flasks were incubated at 30ºC for 7-10 days. After the specified incubation period mycelium was collected by centrifugation at 5000rpm for 10min, and then mycelial free supernatant was used for antimicrobial activity assay.
Extraction of antimicrobial compounds:
The selected actinomycetes were inoculated into 250ml flask containing with previously prepared medium and incubated at 30ºC for 7-10 days. After the specified incubation period the broth was centrifuged at 5000rpm for 10min, and then mycelial free supernatant was extracted with equal volume of ethyl acetate31. Organic extract was further concentrate by evaporation at 41ºC, concentrated extract was mixed with 1ml of ethyl acetate and tested for their antimicrobial activity assay by using Kirby -Baeur well diffusion method32.
Thin Layer Chromatography and Bio autography:
Readymade silica gel pre-coated TLC plates were used for separation of bioactive crude compounds. Dry crude extract was spotted on TLC plate and developed in the solvent system (Ethanol –Ammonia- water/ 8:1:1, v/v). The solvent was allowed to run up to 80% of the TLC plate. Then developed TLC plates were air dried and separated compounds were visualised under UV light and the active spots were detected by bioautography33. Then each spot was carefully scrapped, mixed with ethyl acetate, fraction was collected and was screened against human pathogens.
FT-IR spectroscopic analysis:
FTIR spectrums of Streptomyces isolates were subjected to comparing general pattern, and analyze maximum peaks and functional groups present in the range of wavelength. The Perkine Elmer Spectrum One Fourier Transform Infrared spectrophotometer was used to derive spectrum of each crude extract was determined by potassium bromide (Kbr) matrix with scan rate of 5 scan per minute at the resolution 4cm-1 in the wave number region 450-4000cm-1. The samples were mixed with Kbr, then they were then pelletized by applying pressure to prepare the specimen (the size of specimen about 13 mm diameter and 0.3 mm in thickness) to record the FT-IR Spectrum under Standard condition. FT-IR Spectra were used to determine the presence of the functional groups and bands in the crude extract were observed and interpreted34.
GC-MS spectroscopy analysis:
GC-MS analysis was performed to identify the bioactive compounds present in the crude extract of Streptmyces35. GC-MS analysis was performed on a Shimadzu GC 2010 plus with triple quadrupole mass spectrometer (TP-8030), and fitted with a DB-5ms (5% phenyl methyl siloxane) capillary column of dimensions 30m × 0.25mm × 0.25µm, and with helium as a carrier gas at 1 ml/min. The column temperature was programmed initially at 600C, held for 1.0min, and then increased to 1000C at 5.00C/min, held for 5.0min, and then again raised at 10.00C/min to 2500C, held for 35.0min, and finally raised to 2800C at 10.0◦C/min, and held for 25.0min. The mass spectrometer was operated in electron ionization (EI) mode at 70eV, with an interface temperature of 2800C, an ion source temperature of 2400C, a mass spectrometer acquisition delay time of 3.5min, and a continuous scan from 50 to 650 amu. Peaks were identified by comparison with the mass spectra data from the National Institute of Standards and Technology (NIST) spectral library.
RESULTS AND DISCUSSION:
As a part of our work to search most potent antibiotic producing organism were isolated from vermicast soil. The isolated strains were morphologically identified based on their colour, aerial and substrate mycelium, sporulation pigment, Gram staining and growth of colony in the serial dilution plate. The cultural and morphological characteristics and antimicrobial activity of different Streptomyces isolates were reported by several investigators36. The isolated Streptomyces species showed good antimicrobial activity against Gram -ve and Gram+ve human pathogens. The potent isolated Streptomyces were inoculated in Strach Casein broth containing flask and incubated at 300C for 7-10 days. The optimum temperature for growth is 27-300C and optimum pH is 7.
Organic solvents were tested for extraction of antimicrobial compounds. Organic solvents always play a crucial role to provide higher efficiency in extracting compounds for antimicrobial activity compared to water based methods37, 38. The ethyl acetate extract showed antimicrobial activity against Gram +ve and Gram –ve.
The purification and separation of crude extract compound was done using TLC39. The Rf values was calculated to be 0.73. Then each spot were scrapped carefully and dissolved in ethyl acetate. The collected fractions were subjected to elucidate its structure. The chemical structure of bioactive compounds was studied on the basis of spectroscopic analysis by GC-MS and FT-IR.
The FT-IR spectrum exhibited bands at 3369, 2972, 2932, 2284, 2658, 1648, 1466, 1380, 1341, 1306, 1161, 1129, 1001, 952, 817, 777, 649, 4991 cm-1 from which the presence of amino group, aldehyde , alkanes, alkenes, carboxyl group aromatic, benze, carbon flurene groups was inferred 2425 cm-1. The IR spectrum of the compound included a peak at1466 cm-1 assigned to aromatic C-H cm-1 stretching, whereas the peak appearing at 1446 cm-1 and 1380 cm-1 was assigned to CH3-CH2 stretching (alkanes). The peak appearing at 3369 cm-1 was assigned to OH group (alcohol) stretching. The peak appearing at 1648 cm-1 was C=C alkene group stretching. The peak appearing at 1466 cm-1 was assigned to C=C aromatic group stretching. The peak appearing at 952 cm-1 was assigned to CH3 and the peak appearing at 649 cm-1 was assigned to carbon bromide and peak 491 cm-1 was assigned to iodid.(Fig.1)
GC-MS chromatogram of ethyl acetate extract fractions indicated the presence of different bioactive compounds with different retention time. The chemical structures of compounds are illustrated in figure. GC-MS chromatogram indicated the presence of following six major compounds 12-methyl e-e- 2,13 ocatadecadien-1-ol40 (Fig.2(1)), hexadeconic acid41, (methyl ester) (Fig.2(2)), n-hexadecanic acid42 (Fig.2(3)), 6-octadeconic acid43 (Fig.2(4)), oleic acid44 (Fig.2(5)), octadec-9 –enonic acid45 (Fig.2(6)) including saturated and un saturated fatty acid esters. Some of these fatty acids have been detected from actinobacteria. (Table.1)
Fatty acids act as most promising antimicrobial agents in medicine and the food industry, non-specific mode of action and the lack of resistance mechanisms against the actions of these fatty acids46,47. 9-hexadecenoic acid is the most potent micro antibacterial fatty acid in mice, 6-hexadecenoic acid in humans48. These fatty acids act by destabilising the lipid bilayer of the bacterial membrane. Nonadecane act as component of the bacteriocin produced by a mixed culture of Aeromonas sp. and Enterobacter sp49.
Hexadecanoic acid and hexadecanoic acid methyl ester act as hypocholesterolemic, nematicide, pesticide, lubricant, antiandrogenic, flavour, hemolytic and 5-alpha reductase inhibitor. Other important constituents of GC-MS analysis showed the presence of long chain unsaturated fatty acid (oleic acid ) which are bactericidal to important human pathogenic microorganisms, including methicillin-resistant Staphylococcus aureus50, Helicobacter pylori51, Candida albicans52 and also inhibits the bacterial enoyl-acyl carrier protein reductase (Fab1), an essential component of bacterial fatty acid synthesis which has served as a promising target for antimicrobial drugs.
Hexadeconic acid53 , (methyl ester), n-hexadecanic acid54 (Palmitic acid) has been shown to exhibit potent antibacterial activity have traditional application and effectiveness against such illnesses as skin diseases and respiratory conditions and as anti-inflammatory and analgesics potent mosquito larvicide55. Overall, ethyl acetate extract of Streptomyces species showed six bio active principles identified by GC-MS analysis. According to Lancini et al.56, the activity of an antibiotic is defined and measured in terms of its ability to inhibit microbial growth (bacteria, fungi) and protozoa. These above results showed AS5 which possessed bioactive compounds an attractive strain for further investigation and industrial exploitation.
CONCLUSION:
In the present study, Streptomyces were isolated from vermicast soil, extract of isolated Streptomyces strain showed broad spectrum of antimicrobial activity against Gram+ve and Gram-ve microorganisms. Based on the spectroscopic analysis of extra cellular ethyl acetate extract showed the presence of active principles oleic acid, Hexadeconic acid, (methyl ester), n-hexadecanic acid (Palmitic acid), 6-octadeconic acid. On the basis of the results of this work, the wild isolate of Streptomyces as a good source of bioactive metabolite. It shows promising potential to act as antibiotic for the treatment of human pathogenic bacterial infections.
Table 1. Bioactive constituents identified in the ethyl acetate extract from the fermentation broth of Streptomyces sample (AS-5) using GC-MS analysis
|
Molecular weight |
Compound |
Molecular formula |
Retention time (min) |
|
280.496 g/mol |
12-methyle-e-2,13 ocatadecadien-1-ol |
21.83 |
|
|
270.457 g/mol |
Hexadeconic acid, (methyl ester), |
18.32 |
|
|
256.43 g/mol
|
n-hexadecanic acid (Palmitic acid) |
C16H32O2 |
19.2 |
|
282.468 g/mol
|
6-octadeconic acid |
C18H34O2 |
20.25 |
|
282.47 g/mol |
oleic acid |
C18H34O2 |
21.15 |
|
282.468 g/mol |
octadec-9 –enonic acid |
C18H34O2 |
23.53 |
Fig.1 FT-IR spectrum of Ethyl acetate extract of Streptomyces (AS5)
Fig.2 GC-MS chromatogram of bioactive compounds identified in the ethyl acetate extract from the fermentation broth of Streptomyces sample AS5.
Fig.2 (1) GC-MS chromatogram of 12-methyl e-e- 2,13 ocatadecadien-1-ol
Fig.2 (2) GC-MS chromatogram of hexadeconic acid methyl ester
Fig.2 (3) GC-MS chromatogram of n-hexadeconic acid
Fig.2 (4) GC-MS chromatogram of 6-octadeconic acid (z)
Fig.2 (5) GC-MS chromatogram of oleic acid
Fig.2 (6) GC-MS chromatogram of Octadec-9-enonic acid
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Received on 26.04.2018 Modified on 11.05.2018
Accepted on 27.08.2018 © RJPT All right reserved
Research J. Pharm. and Tech 2018; 11(10): 4569-4574.
DOI: 10.5958/0974-360X.2018.00836.3