INTRODUCTION:

Human mortality has increased due to emerging thrombolytic disorders worldwide (8). A vascular blockage which leads to life-threatening consequences can be caused by a blood clot (thrombus) in blood vessels. The bloodclots in normal blood circulation may suppress the healthy homeostatic system, However, it prevents blood loss by reacting extensively during the vascular injury (5). Thus the failure of hemostasis, the formation of clot inside the circulatory system which cause stroke or myocardial infarction(15). Hence, a conventional treatment for myocardial infarction is usually a thrombolytic therapy, but nowadays prescribed thrombolytic drugs are having side effects like bleeding, re-occlusion and delayed action. (12,13). The clinical interventions for the treatment of suchdisorders are carried out by external administration of thrombolytic agents (2). Streptokinase (SK) is a fibrinolytic agent produced by Streptococcus sp. which is a potential alternative plasminogen activator (3).

There is an increase in the medical significance of the mechanism of the action of SK. Streptokinase activates plasminogen to plasmin due to the formation of 1:1 stoichiometric complex with plasmin this contributes to clot lysis by it proteolytic action on fibrin (4). This increase in potential of streptokinase application prompted us to screen for novel streptokinase-producing organisms. There is also an exponential increase in the applications of streptokinase in different fields during last decades; this increases the demand for both qualitative improvement and quantitative enhancement of streptokinase.

This leading fibrinolytic agent has been included in the WHO Model list of Essential Medicines. It is also used as an intravenous thrombolytic agent in the treatment of acute myocardial infarction. It is known that the fibrinolytic activity of streptokinase enzyme originates from its ability to activate plasminogen (6). The result of the present study is to signify the thrombolytic potential of streptokinase- producing g-hemolytic Streptococci from curd and bore soil samples. This study focuses on different food samples and soil samples for the isolation of potential hemolytic Streptococci and production of streptokinase .

MATERIALS AND METHODS:

Isolation and identification of Streptococcus strains:

Various food samples such as milk, curd, jam, cake, butter and soil samples like bore soil, banana plant soil were collected from various locations in Vellore (Tamil Nadu, India). All the samples were collected in a sterile container and transferred to the laboratory. One gram of soil sample was suspended in 10ml of distilled water and one ml of curd was taken and these samples were mixed thoroughly (gravity cream was removed from curd samples as it may cause contamination of other microbes) and emulsification of the samples was done using 10ml of 1% sodium carbonate solution (freshly prepared) and was kept at 37ºC for 24 h incubation. This prevents the growth of gram-negative organisms(11). These samples were then subjected to serial dilution up to 10^-6dilutions. From dilutions 10^-4to 10^-6 , 0.1 ml of each sample was spread onto nutrient agar plates containing 0.0016% (w/v) sodium azide and were kept for incubation at 37ºC for 24-48 h. The colonies showing the morphological characters like small, pin-point, circular, translucent, convex and moist were selected. Each of selected colonies was cultured on to blood agar plates and pike streptococcal agar plates. The purity of the selected isolates was assured and the strains showing hemolysis on blood agar plates and grampositivecocci in chains were selected for screening.

Screening of Streptococci producing streptokinase:

The isolated colonies were screened based on the growth on casein and human plasminogen in soft agar with slightly modified composition (7). The colonies were plated on-to 20ml of soft agarose mixture that contains 0.8% agarose, 10% skimmed milk, 200µL of human plasma, 150 mMNaCl and 50 mMTris-HCl (pH 8.0). The plates were kept at 37ºC for 12-16 h. The appearance of a zone of clearance around the colonies indicates the streptokinase activity (9). Further, the strains showing a maximum zone of clearance was selected and subjected to various identification processes.

Morphological and biochemical characterization:

The morphology of the isolated colonies was observed. The selected colonies were streaked onto blood agar plates for observing the hemolytic patterns. The potent strains were selected for biochemical characterization. These biochemical tests were performed in reference to Cowan and Steel’sManual for bacterial identification (8).

Production of streptokinase:

The potent strains producing streptokinase were cultured in 100mL of the production medium (g/100 mL: Glucose - 0.5 g, Yeast Extract - 0.5 g, KH2PO4 - 0.25 g, MgSO4·7H2O 0.04 g,NaHCO3-0.1 g, CH3COONa·3H2O-0.1 g, FeSO4·7H2O - 0.002 g, MnCl2·4H2O - 0.002 g, pH 7.5) and kept for incubation at 37°C for 24 h. Once incubation is done, the cultures were centrifuged at 10,000 rpm for 15 min. The cell-free supernatant was passed through 0.45 µm cellulose acetate filter and the filtrates were considered a crude enzyme.

Enzyme activity– Casein digestionmethod:

The potent strains were inoculated in Todd- Hewitt broth and kept for incubation at 37ºC for 18-24 h. The cultures were centrifuged at 4ºC at 8000 rpm for 10 min. The culture supernatant was used as a crude enzyme for the determination of streptokinase activity using casein digestion method, which determines the amount of tyrosine liberated from casein after plasminogen activator.

Partial purification of crude enzyme:

The culture filtrate was precipitated between 40 and 70 % saturation of ammonium sulfate. Using 20 MmTris–HCl of pH 7.5the precipitate was eluted. These elutions were subjected to overnightdialysis against 500 mL of 20 mMTris–HCl at 4ºC.

Determination of total protein content:

The culture supernatant was used as crude enzyme for the estimation of protein content by theBarfords method. The quantity of protein was obtained by plotting graph between concentration of standard BSA and absorbance at 540 nm in a UV-vis spectrophotometer.

In vitro blood clot lysis assay:

The crude enzyme which was extracted from the strains was examined for thrombolytic activity by in vitroblood clot lysis method. Streptokinase (50 µL of 1000 IU/mL) was used as positive control and water was used as negative control (10).

RESULTS AND DISCUSSION:

From various food samples ,curd sample isolate showed γ-hemolysis on blood agar plates and from various soil samples bore soil sample isolates showed β-hemolysis on blood agar plates.(Fig.1) The colonies showed pinpoint, small, circular, moist, convex and translucent morphological characters and were gram positive cocci in chains (Fig.2)and catalase negative.

(a) (b)

Fig 1: Gram staining showing gram-positive cocci in chains: Isolate from (a) Bore soil (b) Curd

(a) (b)

Fig 2: Hemolysis on blood agar plates: isolate from (a) Bore soil(b) Curd

They showed clear zone of hydrolysis on casein plasminogen plates that indicates the maximum streptokinase activity.(fig.3) These selected strains exhibited high activity than other isolates. The isolates showed maximum streptokinase activity and the total protein content was found to be around 15mg in both the samples.

(a) (b)

Fig 3:Casein digestion–Enzyme activity:: isolate from (a) Bore soil (b) Curd

The blood clot lysis activity was visually observed after 3h – 6h of incubation at room temperature. The percentage of clot lysis was quantified. The clot was completely liquefied by 150 µL of enzyme in curd sample and by 250 µL for bore soil sample at room temperature. The crude streptokinase extracted from crude sample showed high activity for casein digestion than that of bore soil sample. (Fig. 4 and 5)

Fig 4: Blood clot lysis (bore soil isolate)

Fig 5: Blood clot lysis (isolate from curd sample)

CONCLUSION:

From the present study, we conclude that streptokinase produced by organisms isolated from curd sample showed high activity than the streptokinase produced by organism isolated from bore soil sample. The results have revealed that the streptokinase activity of curd sample isolates is more than that of bore soil isolates.

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Received on 24.06.2017 Modified on 16.07.2017

Research J. Pharm. and Tech 2018; 11(7): 2845-2847

DOI: 10.5958/0974-360X.2018.00524.3

Screening for Streptokinase producing Streptococcus sp. from Food and Soil samples