Isolation of Melanin Pigment Producing Marine Actinobacterium of Streptomyces Isolated From Marine Sediment Samples and Their Antibacterial Activity

Lakshminarayanan Arivarasu^*, Pravalika Arunkumar and Pitchiah Sivaperumal

^*Correspondence: Lakshminarayanan Arivarasu, Department of Pharmacology, Saveetha Dental College and Hospitals, Saveetha Institute of Medical and Technical Sciences, Saveetha University, Chennai, India, Email:

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Introduction

Melanin is a pigment produced by organisms throughout all domains of life. In the vast majority of studies, melanin has been either chemically synthesized or isolated from animals [1]. In previous researches DOPA melanin showed a promising activity as an antibacterial natural product against 12 pathogenic bacteria from hospital isolations [2]. Currently, the increasing resistance of microorganisms to antibiotics is a serious problem. Actinomycetes, one among the most colourful microbes, are characterized by the production of various pigments on different natural and synthetic media [3]. Marine organisms are the source of thousands of substances, which also have antibacterial and antifungal effects. Microbial mediated production is the best viable alternative to obtain melanin. This method has the advantage of being scalable and providing a good yield of melanin [4]. Color of melanin ranges from yellow to black and depends on the metabolic pathway which synthesises them. Melanins are used in a wide variety of applications in day-to-day life including cosmetics, optical lenses, pharmaceuticals, and batteries to name some [5-10].

In previous studies three strains among 21 Actinomycetes sp isolates produced a diffusible dark pigment on starch casein an agar medium which was water soluble. That study proved that sugarcane waste can be used for the production of melanin and it (melanin) has potential antibacterial activity [11]. A study suggested that, on the basis of anti-bacterial activity and MIC test of melanin pigment from Streptomyces sp., MVCS13 can be selected as an effective antibacterial agent for ornamental fish culture. (‘Melanin from marine Streptomyces sp. (MVCS13) with potential effect against ornamental fish pathogens of Carassius auratus. In an article published it proved that melanin has an antibacterial, antifungal, antioxidant activity and also it can be used for industrial biotechnological application because microbial melanin can be produced in large scale with low production cost [12]. Also few articles proved that actinobacteria isolated from marine environments have been dominated by Micromonospora, Rhodococcus and Streptomyces species. Recent culture-independent studies have shown that marine environments contain a high diversity of actinobacterial species that are rarely, if at all, recovered by cultivation-based methods [13]. Our team has extensive knowledge and research experience that has translated into high quality publications. The present study proves to be unique as not many articles have been published on the antibacterial effect of melanin isolated from marine sediments. The aim of this study is to extract melanin from marine actinobacterium of streptomyces from marine sediments and to analyse the antibacterial activity [14-17].

Materials and Methods

Isolation of actinobacteria

Isolation and enumeration of actinobacteria were carried out in Kuster’s Agar Medium (KUA) supplemented with 0.5% (W/v) NaCl. To minimize the fungal and bacterial contamination, KUA medium was supplemented with cycloheximide (10 µg/ml) and nalidixic acid (10 µg/ml) respectively. Collected sediment samples were serially diluted and inoculated on KUA medium and incubated at 36°C for 7 days. The colonies were counted and the population density has been expressed as colony forming units per gram (CFU/g) of sediments. Morphologically distinct colonies were selected and pure cultures were obtained.

Identification of marine actinobacteria

Aerial mass colour: The colour of the mature sporulation aerial mycelium was recorded in naked eye. When the aerial mass colour fell between two colours series, both the colours were recorded. If the aerial mass colour of a strain to be studied showed intermediate tints, then also, both the colour series were noted. The media used were yeast extract malt extract agar and inorganic salt starch agar [17,18].

Melanoid pigments: The grouping was made on the production of melanoid pigments (i.e. greenish brown, brownish black or distinct brown, pigment modified by other colours) on the medium [19]. The strains were grouped as melanoid pigment produced (+) and not produced (-). In a few cases, the production of melanoid pigments was delayed or weak, and therefore, it was not distinguishable. This is indicated as variable (V). This test was carried out on the media ISP-1 and ISP-7 (Appendix I), as recommended by the International Streptomyces Project [20].

Reverse side pigments: Reverse side pigment production of the isolate was determined on ISP7 medium [21]. The pigment production was noted as distinctive (+) and not distinctive or none (-). In case, a colour with low chroma such as pale yellow, olive or yellowish brown occured, it was included in the latter group (-).

Soluble pigments: Soluble pigment production of isolate was observed on ISP7 medium. The diffusible pigment production other than melanin was considered positive (+) and not produced (-). The colour was recorded (red, orange, green, yellow, blue and violet).

Spore chain morphology: Spore morphological characters of the strains were studied by inoculating a loopful of one week old cultures into solidified agar medium containing sterile glass slides. The cultures were incubated at 28+2°C and examined periodically for the formation of aerial mycelium, sporophore structure and spore morphology [22].

Chemotaxonomical characteristics

Hydrolysis: Hydrolysis was done for releasing amino acids. Harvested cells of each strain weighing 20 mg (fresh) were placed in an ampo bottle and 1 ml of 6N HCl was added and sealed with an alcohol blast burner [23]. The samples were kept at 121°C for 20 h in a sand bath. The bottles were cooled by keeping them at a room temperature of 28+2°C. Hydrolysis was also done for releasing sugars. Harvested cells of each strain weighing 50 mg (fresh) were placed in an ampo bottle and 1 ml of 0.5 N HCl was added and sealed with an alcohol blast burner. The samples were kept at 110°C for 2 h. The bottles were then cooled by keeping them at a room temperature of 28+2°C.

Thin Layer Chromatography (TLC): Spotting of the whole cell hydrolysates was made carefully on TLC plate using a microliter pipette. Spots were 5-10 mm in diameter. This was done by multiple applications on the same spot of very small portions of the sample, which were dried by a hand dryer [24-28].

Amino acids: Each sample (3 µl) was applied on the baselines of the TLC plate (20 cm x 20 cm). Adjacent to this, 1 µl of DL-diaminopimelic acid (an authentic material mixture of DAP isomers) and 1 µl of amino acetic acid (glycine) were spotted as standards [29]. The TLC plate was developed with the solvent system containing methanol: pyridine: glacial acetic acid: H2O (5: 0.5: 0.125: 2.5 v/v). It took approximately more than 4 h for development. The spots were visualized by spraying with 0.4% ninhydrin solution in water-saturated nbutanol, followed by heating at 100 for 5 min. Spots of amino acids ran faster than DAP. The sample spots were immediately compared with the spots of the standards since spots gradually disappeared in a few hours [30].

Whole-Cell sugars: On a cellulose TLC plate (20 cm x 20 cm), 5 µl of samples was spotted along with 3 µl of sugar solutions as standards on the same plates. Galactose, arabinose, xylose and madurose were the sugars, which were used as standards. The TLC plate was developed with the solvent mixture containing ethyl acetate: pyridine: acetic acid: distilled water (8: 5: 1: 1.5 v/v). The development time was more than 4 h. Spots were visualized by spraying with aniline phthalate reagent (3.25 g of phthalic acid dissolved in 2 ml of aniline and made upto 100 ml with water saturated n-butanol). The sprayed plate was heated at 100 for 4 min. Hexoses appeared as yellowish brown spots and pentoses, as maroon coloured spots [31-34].

Assimilation of carbon source: The ability of the actinobacterial strain in utilizing various carbon compounds as source of energy was studied, following the method recommended by International Streptomyces Project [35]. Chemically pure carbon source certified to be free of admixture with other carbohydrates and contaminating materials were used for this purpose. Carbon sources for this test were Arabinose, Xylose, Inositol, Mannitol, Fructose, Rhamnose, Sucrose and Raffinose. These carbon sources were sterilized by ether sterilization without heating. The media and plates were prepared and inoculated according to the convention of ISP project. For each of the carbon sources, utilization is expressed as positive (+), negative (-), or doubtful (±). In the 'doubtful ' strains, only a trace of growth slightly greater than that of the control was noticed [36-38].

Production and purification of melanin

The ISP-2 medium prepared in sea water was used for the development of inoculum. The 2 ml of spore suspension from inoculums medium was inoculated in the fermentation medium containing 0.65 ml of glycerol, 0.63 gm of Yeast extract, 0.55 gm of glucose, .08 gm of MgSO₄.

The 2 ml of spore suspension was inoculated into fermentation medium (ISP7) for 12 days under the agitation for 200 rpm at ambient temperature. Then cell free supernatant was collected by centrifugation at 10,000 rpm for 15 min. The harvested cell free supernatant containing melanin was adjusted to pH 2 with con. HCl and kept at room temperature for 3 hrs. After incubation the suspension was centrifuged at 10000 rpm at 28°C for 20 mins to palletize the melanin pigment. The pellet was washed 3 times with distilled water and dissolved in a phosphate buffer (PH8) [39,40].

Chemical analysis of Melanin

The chemical test was carried out with a little modification. The solubility test for the black pigment was tested by adding 100 µl of melanin pigment in 1 ml of distilled deionized water, 1N HCl, 1M NaOH, absolute ethanol, acetone (warm), chloroform (warm), Phenol and Benzene. The reaction with the following oxidizing agents was also determined by adding 100 µl of melanin to the 1 ml of 30% Hydrogen peroxide (H₂O₂) solution. The precipitation test was carried out by adding 100 µl of purified melanin to 1 ml of 1% Fecl₃ solution and 1 ml of Con. HCl.

Bacterial Suspension: The pathogenic bacterial strains Staphylococcus aureus, Klebsiella pneumoniae and Vibrio cholarae were collected from the Department of Microbiology, Saveetha medical college and hospital, Tamilnadu. The bacterial pathogens were cultured in Muller -Hinton Broth for 24 hr at room temperature. From this bacterial suspension was prepared with saline and the optical density was measured at 600 nm. The concentration of microbial suspension was fixed as 106 CFU/ml. 1 ml of suspension was spread over on Muller Hinton agar plate and incubated for 24 hrs at ambient temperature [41,42].

Antibacterial activity: The antibacterial activity of melanin was performed with a disc diffusion method. Whatman filter paper discs (5 mm) were impregnated with various concentrations (0.5, 1, 1.5, 2, 2.5 and 3 mg/ml) of leaf extract using ethanol and methanol solvent. The inoculated plates were incubated for 24 hr at room temperature and the inhibition zones around the discs were measured. All the results were expressed from an average of three with standard deviation [43].

Minimum Inhibitory Concentration

Minimal Inhibition Concentration of melanin was determined in 5 concentrations (10-5 0 µg/ml/0.001 to 0.1 mg/ml) with blank (extract in Muller Hinton broth). The inoculated bacteria in test tubes are incubated for 24 hr in ambient temperature then the optical density was observed [44-46].

Results and Discussion

Figure 1: The figure shows the isolation and melanin pigment separation.

Table 1: The table shows the cultural characteristics of Actinobacteria. The colour was found to be grey with morphology of the reticulum apertum chain.