Extraction of Actinomycetes from Soil Sediments by Submerged Fermentation: Purification, Characterization and its Anti-Viral Activity.

 

Sumathi R., Ruby S., Krishnakumar Duraisamy and Sivakumar T.

Department of Pharmaceutical Biotechnology, Nandha College of Pharmacy, Erode-52.

*Corresponding Author E-mail: sumoraji@rediffmail.com

ABSTRACT:

Purpose:  The objective of this investigation was to isolate marine actinomycetes, screen them for anti-viral activity and characterize the extract.

Methods: Marine actinomycetes were isolated from sediment samples obtained from various parts of Tamilnadu. The isolates were identified as actinomycetes by microscopical and biochemical tests. Production of active constituents was carried out in two different media, namely, Glucose-Yeast extract Malt (GYM) broth and Luria –Bertani (LB) broth. The extraction of microbial DNA was purified by Agarose gel electrophoresis.

Results:  Among 25 marine isolates subjected to preliminary screening, only isolates S7 and S21 showed potential for anti-viral activity. All two marine soil isolates were synthesized actinomycetes extract with yield ranging from ranging from 32.25 to 53.62 U/ml. However, soil isolates S7 and S21 were selected for further studies because of its high productivity (53.62 U/ml and 52.31 U/ml). The actinomyctes strain are extracted and purified by using agarose gel electrophoresis and compared with marker DNA. The molecular weight of the DNA was determined.

Conclusion:  The study revealed that marine actinomycetes may be potential source of high yield, high substrate specify actinomycetes strain, which is an anti-viral agent.

 

 

INTRODUCTION:

Antibiotics have been used in many fields including agriculture, veterinary and pharmaceutical industry. Marine microbes represent a potential source for commercially important bioactive compounds^1,2. Among marine microorganisms, actinomyctes have gained special importance as the most potent source of antibiotics and other bioactive secondary metabolites^3.

 

True Actinomycetes are bacteria that grow in the form of mycelia, their natural occurrence mostly restricted in the soils. They are gram positive and are related to the coryneform bacteria and mycobacteria by an almost continuous sequence of intermediates forms⁴. Actinomyctes have the capability to synthesize many different biologically active secondary metabolites such as antibiotics, herbicides, pesticides, anti-parasitic and enzymes like cellulose and xylanase used in waste treatment⁵.The actinomycetes derived amphoterin B, also act as antifungal agents.

 

Their role in the treatment of diabetes and also used as antihelmintic agents⁶, anticancer agents, antimicrobials, immunosuppresants. The actionmycetes derived Myxoviromycin, kikumycin and formycin acts as anti-viral agents⁷ and used in the identification of new drug targets by analysis of genomes of pathogens³.

 

EXPERMENTAL:

Materials:

Marine sediment samples were collected from different sites in Tamilnadu, India at a depth of 10cm in March 2009 and placed in new polythene bags, using sterile spatula, and prior to laboratory analysis. Agarose, ά-naphthalene, beef extract, Muller-Hinton agar and Yeast extract were purchased from Hi-media (Mumbai, India). Herpes simplex virus type-I (HSV-1) and Modified eagle’s medium (MEM) were obtained from A.R Laboratories (Salem, India). All other chemicals were of analytical grade and used without further modification.

 

Enrichment and isolation of marine microorganisms:

One gram of sediment was transferred to a conical flask containing 100ml of Glucose-yeast extract malt broth⁵ and Luria-bertani broth⁸ for the pre-enrichment of samples. The flask was incubated at 30⁰C for 14 days in a shaker incubator. A loopful of the inoculums from the pre-enriched Glucose-yeast extract malt broth and Luria-bertani broth was streaked on Glucose-yeast extract malt agar and Luria-bertani agar medium and their plates were incubated at 30^oC for 7 days. Single discrete colonies were isolated and used for identification.

 

Characterization of marine soil isolates:

Isolated colonies were identified using standard International Streptomyces Project (ISP) procedure⁹. Morphological identification of isolated colonies was carried out by simple staining, Grams staining and motility testing by hanging drop method¹⁰. Biochemical characterization was by melanoid production test using Waksman medium at a incubation temperature or 37^oC for 4 days for the detection of pigment producing property of isolates; organic nitrate reduction test was carried out in organic nitrate broth at an incubation temperature of 37^oC for one week for the detection of nitrate reducing property of isolates; acid production test was carried out in glucose nutrient broth at an incubation temperature of 20^oC for 15 days for the detection of glucose fermentation leading to the production of acid; hydrogen sulphide production test was carried out in SIM Agar at an incubation temperature of 37^oC for 5 days ,while gelatin liquefaction test was carried out in nutrient gelatin at an incubation temperature of 37^oC for 24-48 hours for the detection of gelatin hydrolyzing properties of isolates¹¹.

 

Production of crude extract of actinomycetes by submerged fermentation method:

An amount (100ml) of glucose yeast extract malt (GYM) broth (production media, pH 7.0) comprising yeast extract, 0.3gm; malt extract, 0.3gm; peptone, 0.5gm; glucose, 1gm; agar, 1.5gm; water, to 100ml, and contained in a 250 ml Erlenmeyer flask, was inoculated separately with the soil isolates and incubated at 28^oc in a shaker-incubator oscillating at 200 rev/min for 24 hours. At the end of the fermentation period, the crude actinomycetes was prepared by centrifugation at 10,000 rpm at 4⁰C for 10 min. The cell free supernatant was taken as the crude actinomycetes extract¹².

 

Similarly 100ml of Luria-Bertani (LB) broth (production media, pH 7.0) comprised of tyrosine, 1gm; yeast extract, 0.5gm; sodium chloride, 0.2gm; and water, to 100ml was also inoculated with the soil isolates and incubated at 28^oC in a shaker-incubator oscillating at 200 rev/min at 4^oC for 24 hours. The cell free supernatant was taken as the crude actinomycetes extract¹³.

 

Extraction of active compounds:

The resulting eleven supernant were filtered by using whatman filter paper, each supernant was mixed with equal volume of solvents such as ethyl acetate, acetone and methanol. The solvent supernatant mixture was agitated for 45 min, with homogenizer. The solvent was separated from broth by separating funnel; solvent present in the broth was separated by centrifugation at 5000 rev/min for 15 min, to remove traces of fermentive broth. Each extract was evaporated by vacuum desiccators until the dark brown gummy substance was obtained. The crude antibiotic was collected and dried in oven at 40^oC. Residue obtained was subjected to purification¹⁴.

 

Determination of anti-viral activity:

Virus and Cells:

The strain of HSV-1 was propagated in the Vero cells. All cells were grown in MEM with 10% fetal bovine serum.

 

Anti-viral assays:

The anti-viral activities of extracts (S7 and S21) were assayed in 96-well microtiter plates wiyh 30,000 cells/well. The cell culture was incubated at 37^oC for 24 hrs in a humidified 5% CO2 atmosphere. After 24 hrs of incubation, add 100 µg and 50 µg of extracts at dilutions. The cells were incubated for 1 hr. After this period, 50 µl of logarithmic dilutions of viruses were added in triplicate. The plates were incubated again for 96 hrs. Monolayer of cells incubated only with MEM was used as a control. Viral titers were determined by 50% infective doses in tissue culture-TCID50. Antiviral activities were calculated as the difference of virus titer between treated and untreated infected control cultures^15-17.

 

Extraction and Purification of microbial DNA from actinomycetes strains:

2ml of broth (S7 and S21) were taken from each medium (GYM, LB) and centrifuged at 12000 rev/min at room temperature, pellets were collected. Suspended the pellet in to 2ml of sodium chloride buffer and 250 µl of sodium dodecyl sulphate mixed well and incubated at 60^oC for 10 min. Add phenol : chloroform (1:2) mixture, mixed well and centrifuged at 12000 rev/min at 4^oC for 10 min and collected the supernatant. To this add ice cold isoproponol twice the volume  and incubated for 30 min. Followed by centrifuge at 12000 rev/min for10 min at 4^oC and suspended the pellet in 300µl of tris EDTA buffer^17,18. The DNA (Ac01 and Ac02) were collected from the actinomycetes strains.

 

The isolated DNA (Ac01 and Ac02) were        purified by using Agarose gel electrophersis¹⁴. The gel was placed on the gel documentation system under UV- Transilluminator and the bands were obsterved¹⁷.

 

RESULTS:

Enrichment and isolation of marine actinomycetes in different media:

From the 25 marine sediment samples tested, only 11sediments showed growth in enrichment media. The 11 isolates were subculture and used for further studies.

 

Identification of actinomycetes:

All the isolates produced grey and white colonies without pigmentation and showed rapid growth within two days. They were identified as streptomyces spp by slide culture, morphological characteristics, physiological, carbon utilization and nitrogen utilization. The samples of streptomyces species isolated from Tamilnadu were designated S7 and S21. The biochemical characteristics of the isolates are summarized in table1.

Table 1: Biochemical characterization of the isolated organisms.(Note: ‘+’ indicates positive and ‘-‘ indicates negative).

S/NO	Biochemical Characterization	S1	S4	S7	S9	S12	S15	S18	S19	S21	S22	S24
1	Melanoid Formation test	-	-	-	-	+	-	-	-	+	-	-
2	Nitration reduction test	-	-	-	-	-	+	-	-	-	-	+
3	Hydrogen sulphide test	+	+	+	-	-	-	+	+	-	-	-
4	Gelatin liquefacation test	-	+	+	+	+	+	+	+	+	+	+
5	Acid production	+	+	+	-	-	-	-	+	+	+	+

 

Table 2: Anti-viral activity of the actinomycetes strain against herpes simplex virus

S/NO	Extracts	Concentration (µg/ml)	2TCID50	10TCID50	100TCID50
1	Acyclovir	100	+++	++++	++++
		50	+++	++	+++
2	Ac01	100	+++	+++	++++
		50	++	+++	+++
3	Ac02	100	++	+	++
		50	+	+	++

type-1 (Note: ‘+’ indicates 25% inhibition, ‘++’ indicates 50% inhibition, ‘+++’ indicates 75% inhibition, ‘++++’ indicates 100% inhibition. ‘IC’- inhibitory concentration and ‘TCID’- Tissue culture infective dose).

 

 

 

Production of actinomycetes strain:

All eleven isolates examined synthesized actinomycetes extract with yield ranging from 32.25 to 53.62 U/ml. However, soil isolates S7 and S21 were selected for further studies because of its high productivity (53.62 U/ml and 52.31 U/ml).

 

Screening of anti-viral activity:

The anti-viral activity was performed by cytopathic effect (CPE) inhibition assay. The S7 and S21 were obtained from Coimbatore district showed the positive result on Herpes simplex virus type-1 in CPE inhibition assay.

 

Detection of DNA:

The bands of DNA of actinomycetes strain Ac01 and Ac02 were separated according to molecular weight and are compared with marker DNA

 

DISCUSSION:

The search for antibiotics continues to be of extreme importance in research programs around the world because of the increase of resistant pathogens and toxicity of some used antibiotics^19,20. To date, antibiotics are the major bioactive compounds obtained from actionomycetes. However, the ability to produce a variety of antibiotics may attract research interest in these prokaryotes.

 

Among the eleven isolates, the two isolates produce a maximum extracts (32.25 to 53.62 IU/ml).Soil isolates S7 and S21 from Tamilnadu possess anti-viral activity. Among the two media used, the GYM broth shows a better growth of actionmycetes when compared to LB broth. The submerged fermentation shows significant production of actinomycetes crude extracts. By centrifugation, the active components of S7 and S21 were isolated. Using the isolated components, the anti-viral activity was performed by CPE inhibition assay. Acyclovir was used as a standard drug. A comparison of S7 and S21 components shows a significant difference in anti-viral activity. Specifically, S7 shows marked effect when compared to S21.

 

 

In the final purification step, the components were purified by agarose gel electrophoresis method, the DNA (Ac01 and Ac02) bands were observed by comparing with marker DNA. The two DNA bands showed moderately equal molecular weight.

 

CONCLUSION:

From the present study, it is concluded that marine sediment samples from Tamilnadu in India are potential sources of bioactive actinomycetes but also demonstrates that different pretreatment methods may be successfully applied for the isolation of actinomycetes. The isolated actionmycetes strain shows very good inhibition over the virus Herpes simplex virus type -1. This was used for developing a new drug against such type of virus, which may be very safe, less toxic and cheap when compared with other drugs. Consequently the results indicates with confident from the present study will render satisfactory results for further studies.

 

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Received on 19.02.2010       Modified on 21.03.2010

Accepted on 15.04.2010      © RJPT All right reserved