EXTRACTION AND ESTIMATION OF PIGMENTS FROM BACTERIA

 EXTRACTION AND ESTIMATION OF PIGMENTS FROM BACTERIA 

AIM: 

The current study is focused on antibacterial pigment production from Yercaud hills region soil bacteria. 

PRINCIPLE:

 ● In this present study, a soil sample was collected from a coffee plantation in Yercaud, Salem district. The collected soil sample was used 

● To isolate the pigment-producing bacterial strains. The pigments were extracted by acidified ethanol and there used to screen the antibacterial 

● Activity against clinical pathogens. The potential antibacterial pigment producer was identifying and the pigment was characterized by ultraviolet 

● Spectroscopy, thin-layer chromatography, and Fourier Transform Infrared (FT-IR) and also evaluate the minimum inhibitory concentration of pigment

 ● Against clinical pathogens. 

PROCEDURE: 

METHODS: 

Collection of Sample: 

● The soil samples were collected from rhizosphere of coffee plant area In Yercaud, Tamil Nadu From all samples, pigment-producing bacteria 

● Were isolated and the bacterial cultures were used for further study. 

● Isolation of pigment-producing bacterial strains

● Collected soil samples were used for serial dilution up to 10−7 dilutions. 

● A 0.1 ml of aliquots from 10−4 to 10−6 dilutions were spread on nutrient 

● Agar plates and the plates were kept for incubation at 37°C for 48 h. 

● After the incubation, nutrient agar plates were observed for growth and 

● Pigment production. Pigment-producing bacterial isolates were used 

● For further study

Extraction of pigment from pigment-producing bacteria:

● The pigment-producing bacterial strains were harvested by centrifugation at 6000 rpm for 10 min 

● Then, the supernatants were discarded and the pellets were resuspended in acidified ethanol (4 ml 

● Of 1 M HCL in 96 ml ethanol) 

● Then, the mixture was vortex and the suspension was centrifuged at 6000 rpm for 10 min and the supernatant Was collected. 

● The centrifugation was repeated till the pellet changes To colorless. 

● The crude pigments were allowed to acidified ethanol evaporation.

 ● After the evaporation of acidified ethanol, dried pigment Was collected for further uses. 

Screening of antibacterial pigment-producing bacteria 

● An overnight log culture of each pathogenic strain such as Escherichia Coli, Klebsiella sp., Enterococcus sp., Pseudomonas sp., and Proteus sp. was spread evenly on a Mueller-Hinton agar (MHA) plate by swab. 

● Wells were made on the MHA plates using a gel puncture. 

● After that, the extracted crude pigment (100 µl) was added into each well, and the plates were incubated at 37°C for 24 h. 

● After incubation, the zones of inhibition were measuring the diameter of the zone formed around each well.

 Identification of potential pigment-producing bacteria 

• Colony morphology (size, shape, color, margins, opacity, consistency, And elevation) Gram staining and motility characterization of pigment-Producing bacteria were done. 

● The biochemical characteristics were Found by various biochemical tests such as indole, methyl red, Voges–Prostkauer, citrate utilization, urease, oxidase, catalase, and triple sugar Iron test. The pure culture of isolate was maintained on nutrient agar Slant for further investigation

Characterization of extracted bacterial pigment 

● Ultraviolet (UV) spectroscopy analysis of crude pigment 

● Maximum absorption spectra of the obtained bacterial pigment were performed using UV spectrophotometer.

 ● UV spectrum analysis of the obtained bacterial pigment (1 mg/ml) was suspended in a 50 mM Tris-HCl (pH 8.5) solution in the wavelength range between 350 and 750 nm to find out the maximum absorption spectra and Tris-HCl 50 mM (pH 8.5) as blank. Thin-layer chromatography (TLC) analysis 

● The purified pigment was analyzed by TLC with silica gel. The solvent system consists of chloroform: methanol (9:1; v/v). 

● The chromatography Chamber with the solvent was kept for 20 min for the equilibration. 

● The Sample was spotted on the silica gel sheet using a capillary tube and Air-dried.

 ● The TLC sheet was then dipped in the solvent system. After 45 min, the TLC sheet was carefully removed and the retention factor (Rf) value was calculated according to the following equation from the Chromatogram. 

By the compound Rf = Distance travelled by the solvent  

                                         Distance travelled by solvent 

Fourier-transform infrared (FT-IR) spectroscopy analysis 

● The pigment was analyzed using FT-IR spectroscopy. The crude Bacterial pigment was encapsulated in KBr at a ratio of 1:100. The IR spectra were collected using a shimadzu spectrometer within the rof 4000– 400 cm−1. 

● The FT-IR spectroscopy is used to analyze the functional group of the extracted pigments. 

Minimum inhibitory concentration (MIC) of bacterial pigment 

● The clinical pathogenic (E. coli, Klebsiella sp., Staphylococcus aureus, Pseudomonas sp., Proteus sp., and Enterococcus sp.) cultures were inoculated into the nutrient broth and incubated for overnight. 
● The MHA medium was prepared and the petri plates were sterilized. Then, sterilized medium was poured into the plates and allowed them to solidify. The clinical pathogens were swabbed onto the plates. 

● Well was Cut in the agar plates using well puncture. The extracted crude pigment was dissolved in acidified ethanol and 20 µl, 40 µl, 60 µl, 80 µl, and 100 µl were added to the well and incubated these plates at 37°C for 24 h.  

RESULTS: 

Isolation of pigment-producing bacteria 

● The collected coffee rhizosphere soil samples were subjected to spread plate method on nutrient agar plates. 

● The bacterial cultures were Selected from based on the different colony morphology. A total of 30 bacterial strains (YP1–YP30) were isolated from soil sample. 

● Among the 30 bacterial isolates, four isolates (YP1, YP3, YP5, and YP27) were producing pigments and the pigmented bacterial colonies were Used to further screening process.

Screening of antibacterial pigment-producing bacteria 

● The potential pigment-producing four bacterial isolates were centrifuged and collected; the cell-free supernatants were used to Screening as a crude pigment. 

● Among the four pigment-producing Isolates, the isolate YP1 showed maximum zone of inhibition against 

● Test clinical pathogens. 

● The highest activity producing YP1 Bacterial strain was used as further analysis. 

Identification of the potential pigment-producing bacterial isolate 

● The bioactive potential pigment-producing bacterial isolate was Then preliminary characterized, according to Bergey’s Manual of Determinative Bacteriology based on morphological and biochemical Characterization.

 ● The antibacterial pigment-producing bacterial isolate Was translucent, smooth colonies of about 1 mm diameter with profuse Brown pigmentations were selected for further studies. 

● The biochemical Characterization of the potential bacterial isolate is tabulated .

 ● The isolated potential bacterial strain was identified as Roseomonas Gilardii by 16S rRNA sequencing. 

● The isolated antibacterial pigment-producing bacterial isolate YP1 showed 98% similarity with R. gilardii and their GenBank accession number is MH324463. 

Extraction of bacterial pigment 

● The antibacterial pigments were extracted from YP1 pigment-producing Bacterial strain and their extracted pigment was characterized For further analysis. 

UV–visible spectroscopy analysis of crude pigment 

● Absorption spectra of pigments from YP1 strain were studied in the visible range between the wavelength range of 350 and 750 nm and spectrophotometric analysis at the respective wavelength at which maximum absorbance was absorbed at 450nm.

TLC analysis of crude pigment 

● The extracted pigment was separated by TLC with silica-coated TLC Plate. The solvent system of chloroform: methanol (9:1) was used for The separation of pigment. TLC showed the presence of a pigment Which migrated as brown component in the sheet with Rf 0.82.

 FT-IR analysis of crude pigment 

● The extracted crude pigment was characterized by FT-IR spectrum. Based on the FT-IR spectra, pigment had containing the carboxylic Acids, alkenes, phenols, alkanes, and primary amines functional groups. 

Minimal inhibitory concentration of pigment against clinical Pathogens 

● The pigment was tested for antibacterial activity against six selected Clinical pathogens such as S. aureus, Klebsiella sp., Enterococcus, E. coli, Pseudomonas sp., and Proteus sp. By well diffusion method. The Maximum zone of inhibition observed in E. coli bacterial strain. 

DISCUSSION:

 ● A total of 30 morphological different bacterial strains were isolated from the coffee plant rhizosphere soil sample in Yercaud hills region, Tamil Nadu

● Among the 30 bacterial strains, only four bacterial strains Producing antibacterial pigments. However, the brown pigment-Producing colonies were found predominant. Earlier studies in 15 pigment-producing bacteria were isolated from eight soil sample

CONCLUSION

● The polyphenolic rich coffee plant rhizosphere constitutes have Extremophilic condition in addition to the lower atmospheric Temperature prevailing in Yercaud that greatly influences bio-geo Nutrient recycling by retarding. Extremophiles have the tendency. To acquire potential characteristics for their survival in competitive Environment. 

● Hence, the presence study focused on pigment-producing Bacteria from extremophilic condition and found a higher pigment-Producing activity with potential antimicrobial characteristic in the Isolates. 

● This study thus attempted to explore the extremophilic Microbial ecology for pigment production with its antimicrobial Characteristics. 

● The identified isolates from soil were found to the YP1 pigment strain, able to produce pigment. In this regard, this study Is an initiative approach toward the use of biocolorants which find Its applications in numerous sectors as an alternative for synthetic Chemicals. 

● To conclude, microbial pigment production is one of the Emerging fields of research to demonstrate its potential for various Industrial applications