INTRODUCTION:

Natural pigments are used because of the strong consumer demand for more natural products. The accessible authorized natural pigments from plants have numerous drawbacks such as instability against light, heat or adverse pH, low water solubility and are often non-availability throughout the year^[1]. The bio pigments can be obtained from 2 major sources plants and micro organism. Bio pigments from microorganism are preferred than plants because of their stability cultivation technology. Hence, microbial pigment production is now one of the emerging fields of research to demonstrate its potential for various industrial applications^[2].

Serratia species:

Serratia genus is a member of the enterobacteriaceae family is a gramnegative bacterium belonging to the serratia genus, organism is recognised for its production of a bright red pigment called prodigisian. Optimum growth of all strains of serratia has been observed at pH 9 and at temperature from 20-30⁰C degree environmental. Serrtia sp isolated from infected adults generally does not produce prodigiosin^[3].

Prodigiosin a natural red pigment with family of tripyrrole characterized by pyrrolylpyrromethane skeleton. Prodigiosin has an usual structure with three pyrrole rings and pyrryldipyrrylmethane, two rings are linked to each other and third is attached by a way of methane bridge. There are four classes of prodiginines. The first group consists of prodigiosin with straight chain alkyl substituents such as prodigiosin and undecylprodigiosin. The other three groups contain cyclic derivatives of prodiginines.^[4]

Figure:1 Structure of prodigiosin

METHODS:

Sample collection:

Sample of serratia species were provided by Department of Microbiology, Dr. MGR Education and Research Institute University, Velapanchavadi, Tamilnadu.

Production of biopigments:

Nutrient agar slant were prepared and inoculated. The test tube were incubated at 30⁰C for 24hrs. Incubation was carried out under static condition. After the development of the microorganism the test were stored in refrigerator for futher studies^[5]. The microorganisms in slant was taken and inoculated in each and every conical flask which contain broth medium. After inoculation the conical flask are incubated at 28⁰C for 72 hours in shaking incubator or water bath shaking incubator at 75 rpm.

[OD₄₉₉– (1.381x OD₆₂₀ )]

Prodigiosin =------------------------------------- x 1000

unit/cell OD₆₂₀

Where,

OD₄₉₉– pigment absorbance

OD₆₂₀– bacterial cell absorbance

1.381 - constant

Estimation of pigment:

The 10ml of nutrient broth was taken in centrifuge tube. It was subjected to cooling centrifugation at 15,000 rpm for 15mins at 4⁰C^[6]. The above was divided as supernatant and cell pellets. To the cell pellets add 10ml of 95% acidified methanol and centrifuge under same condition. Debris was removed and the 2ml of the supernatant was taken in two test tubes. The content of one of the test tube was acidified with a drop of concentrated HCL and the other alkalinized with a drop of concentrated ammonia solution^[7]. A red or pink colour in the acidified solution and a yellow to tan color in the alkaline solution indicates a positive presumptive test for prodigiosin. 5ml of the supernatant was subjected to spectrum scanning in the range of 300-700 nm using UV-VIS spectrophotometer. 95% methanol was used as a blank. Methanolic extract of prodigiosin showed characteristic maxima at 499nm^[8].

The results were given in Graph No.1 and 2.

Factorial Design:

Physical Parameters:

Effect of Temperature, pH and RPM^[5,9]:

Incubation temperature is one of the important parameters which helps in the growth of the micro organisms, temperature even plays the role in the pigment production as some of the micro organisms have proved to produced pigment only at particular temperature. The isolates were inoculated in broth and incubated at different temperature like 25, 28, 30 and 37⁰C for 3 days, having different ph such as 6.5,7, 7.5 and 8 with different rpm like 50, 75 and 100.

The results were given in Graph No.3, 4 and 5.

Screening of Carbon and Nitrogen sources:

Effect of carbon sources^[10]:

Serratia marcescens demonstrated the highest pigment production (560 unit/cell) among the three examined strains with crude glycerol as carbon source. Among the four tested carbon sources, crude glycerol displayed easy prodigiosin recovery due to its liquid state which did not absorb part of the produced pigment. The addition of the ﬁve tested nitrogen sources to crude glycerol revealed that peptone. The results were given in Graph No.6 and 7.

RESULTS:

The results were below,

Graph No.1: Yield of Produced Prodigiosin

Microorganism A2 was found to produce more drugs/unit cell,

which was (A2) was taken for further studies.

Growth curve:

The cultured microorganism were collected periodically in 12 hrs gap and optical density was absorbed. The graph was plotted against hours and O.D value.

Graph No.2: Growth pattern of Produced Prodigiosin

Physical Parameters of Produced Prodigiosin:

Graph No. 3: Effect of Temperature

Graph No. 4: Effect of pH

Graph No. 5: Effect of RPM

Carbon and Nitrogen sources:

The carbon sources and Nitrogen source were used separately. The maximum yield was found to be,

Graph No. 6: Optimization of Carbon sources

Nitrogen sources:

The nitrogen sources used were L-proline, cystine, peptone and ammonium chloride. The maximum yield was found to be,

Graph No. 7: Optimization of Nitrogen sources

CONCLUSION:

With the tough demand to search for novel compounds, prodigiosin is a bio – metabolite that owns various medically potent anticancer and immunosuppressant with reported positive eﬀect on the increased chances of survival in mice undergoing heart transplantation^[11]. Glucose a waste obtained from biodiesel industry was used to support growth of Serratia marcescens A2, giving the highest prodigiosin production among the tested carbon sources due to its unique composition where it contains glycerol, methanol, soap, water and trace amounts of ions (mainly Na+ and K+) in variable percentages depending upon the method of production. The production studied are analysed by measuring the optical density produced and it reflect that 1% of glucose carbon source given remarkable yield^[12,13]. In nitrogen source, 0.25% of peptone showed phenomenal pigment production.

REFERENCE:

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11. Siva R, Subha K, Bhakta D, et al. Characterization and enhanced production of prodigiosin from the spoiled coconut. Appl Biochem Biotechnol. 2012, 166:187–196.

12. Mathivanan N (1995) Studies on extracellular chitinase and secondary metabolites produced by Fusarium chlamdosporum, an antagonist to Puccinia arachidis, the rust pathogen of groundnut.

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Received on 20.02.2019 Modified on 15.03.2019

Research J. Pharm. and Tech. 2019; 12(7):3475-3478.

DOI: 10.5958/0974-360X.2019.00589.4