Introduction
Plants and microorganisms are the two major sources to obtain natural pigments. The natural pigments obtained from plants have numerous drawbacks such as seasonal availability, instability against light, heat, pH and low water solubility.1 Pigment production from microorganisms includes easy and fast growth in culture medium and it is independent from weather conditions.2 Prodigiosin is a secondary metabolite produced by various microorganisms such as Serratia, Streptomyces, Pseudomonas,Pseudoalteromonas sp. having a common pyrrol dipyrromethane core structure.3 Serratia marcescens is a single short rod bacteria belonging to the family Enterobacteriaceae, which is a gram negative, motile and facultative a naerobic in nature. Prodigiosin has a tripyrrole in its structure belongs to the family prodiginines. The production of prodigiosin is susceptible to temperature and is significantly inhibited at a temperature higher than 37◦ C in Serratia marcescen and utilized to produce antibacterial spray.
Materials and Methods
Prodigiosin production grown on MacConkey agar
Serratia strain was extracted from the soil and streaked on MacConkey agar plate. The plate was placed in room temperature for 48 hrs. After 48 hrs, the plate was observed to have yellow coloured colonies. These colonies were inoculated into the LB broth and placed in CO2 incubator under room temperature for 48 hrs. After 48 hrs, the prodigiosin pigment was observed.
Prodigiosin production grown on Luria Bertani agar
Serratia strain was extracted from the soil and streaked on Luria Bertani agar plate. The plate was placed at room temperature for 48 hrs. After 48 hrs, the plate was observed to have pinkish red coloured colonies. LB broth was taken and pinkish red coloured colonies were inoculated into the LB broth and placed under room temperature for 48 hrs. After 48 hrs, the prodigiosin pigment was grown in the broth.
Extraction of pigment grown on MacConkey agar
Serratia marcescens in MacConkey agar was centrifuged at 8000 g for 30 min. The Cell debris was formed in low concentration.
Extraction of pigment grown on Luria Bertani broth
Serratia marcescens in Luria Bertani broth was centrifuged at 8000 g for 30 min. The supernatant was discarded and the cell pellets were collected. The acidified methanol was added with the extracted cell pellet and was centrifuged at 5000 g for 15 min. The supernatant was collected in a tube and placed in a dry bath at 60◦C for 48 hrs to obtain a crude pigment.
Result and Discussion
Estimation of prodigiosin from LB agar
The cell absorbance of the bacterial culture was measured at 620 nm. For the pigment absorbance, the broth was mixed with methanol, subjected to centrifugation. The supernatant was used for the measurement of absorbance at 534 nm. The pigment prodigiosin was estimated by the formula,4
The pigment prodigiosin was estimated by the following formula and it was found to be 36.9 units/cell.
OD534 = pigment absorbance; OD620 = bacterial cell absorbance; 1.381 = constant
Microbial strain
Escherichia coli and Pseudomonas sp.were used for the evaluation of antibacterial activity.
Nutrient agar
Nutrient agar was prepared (peptone -2.5 g, yeast -1.5 g, distilled water-500 mL, agar-10 g), stirred, boiled to dissolve and autoclaved at 121◦C for 15 min. The hot medium was poured in a sterile petriplate. The medium was allowed to solidify for 15 min.
Agar well diffusion method
The antibacterial activity of the pigment was carried out using agar well diffusion method separately. The test bacteria such as E. coli and Pseudomonas sp.were inoculated on the nutrient agar plate and incubated at 37˚C. 20 mL of methanolic extract of this pigment was impregnated on the surface of test bacteria. Wells were made to inoculate the extracted pigment in different proportions on the surface of E.coli and Pseudomonas sp. The clear zones of inhibition were observed by incubating the plates at 37◦C for 36 to 48 hrs.5
The results of extraction of prodigiosin from Serratia marcescens and demonstration of antibacterial properties on two bacterial isolates, obtained in this study, imply that the growth rate of the prodigiosin pigment is higher in LB agar when compared with MacConkey agar. Incubation of MacConkey agar plates under increased CO2 has been observed to reduce growth of microorganism. The pigment from MacConkey agar was obtained in lower concentration. The antibacterial activity of the pigment from LB agar was estimated and checked by well diffusion method on E.coli and Pseudomonas sp. in different concentrations. Demineralized water was mixed with the dry extract and then lavender essential oil was added in the mixture which has characteristics of antibacterial, antifungal activities and it has a pleasant smell. This indicates that, it can be useful to remove unwanted microbes in glassware which are used in laboratories and industries.
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