Nature is rich in colours (minerals, plants etc.) and pigment producing microorganisms (fungi, yeasts and bacteria). Microbial pigments such as carotenoids, melanins, flavins and quinines are used in many food industries (Dufosse, 2006). The ability to synthesis pigments varies with the organism and its environment. Fluorescent Pseudomonads are ubiquitous bacteria that are common inhabitants of the water, soil, plants and animals and they are the most studied group.
Pseudomonas aeruginosa produces variety of extracellular pigments, of which phenazines comprise a significant portion. Phenazine compounds produced by fluorescent Pseudomonas species are biologically active metabolites that function in microbial competitiveness (Mazzola et al., 1992). Strains of P. aeruginosa produce variety of redox-active phenazine compounds, including pyocyanin, phenazine-1-carboxylic acid and phenazine-1-carboxamide (Budzikiewicz, 1993). Pyocyanin is the main phenazine pigment associated with this particular organism. About 90 to 95% of P. aeruginosa strains produce pyocyanin (Smirnov and Kiprianova, 1990). Defined conditions for the reliable production of phenazines have greatly aided by biosynthetic studies (MacDonald, 1967). The simultaneous accumulation of more than one phenazine by P. aeruginosa had been reported (Korth et al., 1978) and culture conditions which differentially affect the fractional composition of phenazine pigments had also been described (Kanner et al., 1978). Pyocyanin inhibits Nitrous Oxide (NO) synthesis and blocks the relaxation of rabbit aortic smooth muscle induced by NO and organic nitrates. Pyocyanin has a variety of pharmacological effects on eukaryotic and prokaryotic cells (Vukomanovic et al., 1997).
P. aeruginosa secretes proteins which are toxic to a wide range of organisms and it also secretes copious amount of phenazine pigments (Chang and Blackwood, 1969). Phosphate depletion within this microbe has been suggested to be the initiator of phenazine biosynthesis. Pyocyanin interferes with the regulation of ion transport, ciliary beat frequency and mucus secretion in airway epithelial cells by altering the cytosolic concentration of calcium (Denning et al., 2001). Pyocyanin is known to suppress wheat blotch caused by Septoria tritici (Flaishman et al., 1990). Phenazines compounds produced in the rhizosphere of plants contribute to the biological control activity of P. aeruginosa against Fusarium wilt of chickpea and Pythium damping of bean (Anjaiah et al., 1998). Due to the abundant occurrence and considering the biotechnological application of P. aeruginosa the present study was under taken with the following objectives: isolation and identification of Pseudomonas aeruginosa from marine environment, estimation of pyocyanin and pyorubrin pigment production. Application of pigments as food colourants and antibacterial effect of the pigments against pathogenic bacteria.
MATERIALS AND METHODS
Isolation and Identification of P. aeruginosa
Sediment samples were collected in the mangrove environment from Vellar
estuary (Lat. 11° 29N; Long. 79° 46E) using sterile spatula
and aseptically transferred in to sterile polythene bag and further analysis
was carried out in Centre of Advanced Study in Marine Biology, Parangipettai,
Tamil Nadu, India during January to March 2007. The sediment samples were serially
diluted and plated on Zobell Marine agar and incubated at 35°C for 48 h.
All the blue green pigmented colonies were purified on Zobell Marine agar and
stored in slants with same medium at 4°C until use. Identification of P.
aeruginosa was done based on morphological, cultural, biochemical and physiological
characteristics as suggested by Cappuccino and Sherman (1999) and Schaad et
al. (2001) and the results were crosschecked with Bergeys Manual of
Determinative Bacteriology (Buchanan et al., 1974).
P. aeruginosa was grown in the medium described by Frank and DeMoss
(1959). The mineral salt medium contains (g L-1 until other wise
specified) DL-Alanine-10, Glycerol-20 mL, K2HPO4-0.139,
MgCl2.6H2O-4.06, Na2SO4-14.2, Ferric
Citrate-0.1, 50% seawater-1000 mL and pH 7±0.02. Sterilized medium was
inoculated with 1.0% of 18 h culture and incubated at 35°C for 72 h.
Extraction, Estimation of Pyocyanin and Pyorubrin Pigments and Biomass
Two volumes of chloroform was added to one volume of cell free culture supernatant
and shaken well. The pyocyanin was then extracted from the chloroform into 0.2
N HCl to this deep red acid solution 0.4 M borate-NaOH buffer (pH 10) was added
until the colour changed to blue and the blue coloured pyocyanin was again extracted
into chloroform. This step was repeated 2 or 3 times, resulting in a clear blue
solution of pyocyanin in chloroform and pyocyanin powder was collected by evaporating
the chloroform (Frank and DeMoss, 1959). After the extraction of pyocyanin in
chloroform phase, pyorubrin in the aqueous phase was separated and lyophilized
A standard pyocyanin and pyorubrin pigment graph was prepared with known concentrations of pigments. Concentration of pigments in the culture broth was estimated by measuring the OD at 520 nm and the obtained OD values were compared with the standard graph and the concentration of the pigments were expressed as mg mL-1.
Broth culture samples collected at different intervals were centrifuged at 10,000 rpm for 15 min to obtain the cell pellet. The cell pellet was dried in hot air oven at 80°C for a period of 24 h and the biomass was quoted in terms of mg mL-1 (dry weight).
Pigments as Food Colourants
Various concentrations of pyocyanin and pyorubrin were prepared (5, 10,
15, 20 and 25 mg mL-1) and 3% of agar was added to the pigment solution
and heated to boiling and then cooled for solidification. This test was made
to ensure the reliability of the pigments and their colouring ability at boiling
temperature in food materials.
Antibacterial assay was carried out by well diffusion technique for each
pigment along with a control i.e., chloroform for pyocyanin and distilled water
for pyorubrin. Wells with 0.5 cm diameter were made on sterile Muller Hinton
agar plates. Pathogenic bacteria namely Salomonella paratyphi, Escherichia
coli, Citrobacter sp. and Klebsiella pneumonea were swabbed
on the surface of the agar and different concentration of pyocyanin/pyorubrin
pigments (5, 10, 15, 20 and 25 μL) were added in to the wells. Plates were
incubated at 37°C for 24 h. Antibacterial activity of the pigments was determined
by measuring the growth inhibition around the well.
Hemolytic Activity of Phenazine Pigments
Fresh sheep blood was collected from a nearby slaughter house and EDTA (2.7
g in 100 mL distilled water) was added as anticoagulant at the rate of 5% of
the volume of blood. The blood was centrifuged at 5000 rpm for 7 min at 4°C
along with normal saline and the supernatant was discarded. 1 mL of the packed
RBC thus obtained was resuspended in normal saline to obtain a 1% RBC suspension.
The same procedure was adopted for chicken blood also.
The assay was carried out in Laxbro microtitre plates. The lyophilized pyorubrin and the air dried pyocyanin pigments were assayed. Various concentration of the pigment was prepared. One row of well was used for only one concentration. Initially 100 μL of normal saline was added to each well. Then 100 μL of the lowest concentration of the pyocyanin pigment was added to the first well and this process was repeated up to the last well from which 100 μL was discarded. Then 100 μL of the prepared erythrocyte suspension was added to each well.
A negative control was kept by mixing 100 μL of normal saline and 100 μL of 1% RBC suspension. Formation of a fine button cell with regular margin indicates the negative reaction. A uniform red coloured suspension of the lysed RBC indicates the positive result. The plates were incubated for 3 h at room temperature and the hemolysis was observed.
Isolation and Identification of P. aeruginosa
Out of 31 different colonies isolated in Zobell marine agar medium,
only two strains showed luxuriant growth in Pseudomonas isolation medium
and also produced blue green diffusible pigment. These two isolates were subjected
to morphological, cultural, biochemical and physiological characterization.
Based on the results the isolates were identified as Pseudomonas aeruginosa
(Table 1). Out of the two strains producing pigments,
one strain was chosen for the present study as it showed comparatively intense
Pigment Production and Characterization
Pigment production in mineral salt medium revealed that, the appearance
of pigment starts at 6 h in the culture medium. The pyocyanin and pyorubrin
was estimated at 6 h intervals. Pigments showed steady increase in concentration
through out the incubation period. The initial pyocyanin production was comparatively
more than pyorubrin. The initial pyocyanin pigment concentration at 6 h was
3.50 mg mL-1 whereas the initial concentration of pyorubrin was 2.50
|| Physiological and biochemical characteristics of P.
|+: Positive, -: Negative
|| Pyocyanin and pyorubrin production by P. aeruginosa
There was a correlation observed between the biomass and pigment production
up to 66 h after which decrease in biomass concentration was observed (Table
Pigments as Food Colourants
In pyocyanin, all the concentration showed good, pleasant colouration. The
maximum intensity was observed in 25 mg mL-1 (Fig.
1) and the least colouration was observed in 5 mg mL-1 with pyocyanin.
Even in lower concentration the colour obtained with the pigments was pleasant
to see. Like wise pyorubrin showed a chocolate brown colour and other things
holds good with that of pyocyanin.
Antibacterial Activity of Pyocyanin and Pyorubrin
Pigment extracts from P. aeruginosa showed distinct antibacterial
activity against Citrobacter sp. Antibacterial activity was found
in all the concentrations (5, 10, 15, 20 and 25 mg mL-1) of pyocyanin
and pyorubrin pigments. Lower concentrations of both the pigments showed lower
inhibitory zone when compared to the higher concentration. The maximum inhibition
zone was found with pyocyanin pigment (1.7 cm) and the minimum inhibition zone
was observed with pyorubrin pigment (1 cm) (Table 3).
|| Antibacterial activity of pyocyanin and pyorubrin against
|| Hemolytic activity of pyocyanin and pyorubrin pigments
|| Pyocyanin (A) and pyorubrin (B) mixed with agar
Pyocyanin and pyorubrin pigments were found to be hemolytic positive for
the concentration of 15, 20 and 25 mg mL-1 (Table 4)
in chicken blood. There was no hemolytic activity at 5 and 10 mg mL-1
concentration of both the pigments. With sheep blood no hemolytic was observed
even in higher concentration i.e., 25 mg mL-1.
Isolation of P. aeruginosa was carried out in Zobell marine agar. Out of 32 colonies, only 2 colonies (6.25%) showed intense blue green pigmentation and identified as P. aeruginosa. The strain which showed intensive pigment production was used for further study. The strain produced two pigments, one with blue colouration and another with greenish brown colour named respectively as pyocyanin and pyorubrin.
Regarding pigment production, the pyocyanin and pyorubrin pigments showed a
steady increase in concentration throughout the culture period (72 h) and these
results were comparable with that of Graham et al. (1979). In the present
study, the stability of pyocyanin pigment concentration after 60 h may be due
to the range of phenazine compounds produced in the growth phase or in response
to environmental signals. The biomass showed its maximum at 66 h and attained
declined phase at 72 h, the close association between growth and pigment production
from 6 to 66 h effectively showed the utilization of substrate for growth as
well as for pigment production.
Both the pigments of P. aeruginosa showed distinct antibacterial effect against Citrobacter sp. However greater effect was found with pyocyanin pigment. Production of antibiotics by P. aeruginosa has been recognized as a major factor in controlling many pathogens. The phenazine compounds are phenolic in nature, which includes pyocyanin and pyorubrin pigments, exhibit broad spectrum antibiotics. Phenazines are synthesized from chorismic acid, possibly via anthranilate, which is also an intermediate in the tryptophan biosynthesis pathway (Essar et al., 1990). Anthranilate is synthesized from chorismate by two distinct anthranilate synthases in P. aeruginosa. Phenazine B, whose deduced amino acid sequence is similar to those of anthranilate synthases, required for phenazine antibiotic production in P. aureofaciens (Pierson and Pierson, 1995). P. aeruginosa PNA1, when grown in culture, produced phenazine antibiotics phenazine-1-carboxylic acid and oxychloraphine and inhibited the mycelial growth of F. oxysporum, P. splendens and other phytopathogenic fungi (Anjaiah et al., 1998). Citrobacter a member of the family Enterobacteriace, may cause urinary tract infections, wound infections and some times pneumonia in humans especially in immunocompromised persons (Hodges et al., 1978). In urinary infection Pseudomonas sp. and Citrobacter sp. co-exit in this regard the present result may be valuable.
The idea of checking the pigments antibacterial activity against water and food borne pathogens was whether it has an additional protective effect when used as a food colourant. Results obtained in the present study were not encouraging, because the pigments showed antibacterial activity only against Citrobacter sp. However this cannot deter the pigments in using them as colourants.
Hemolytic assay inferred that both the pigments did not show any hemolytic activity at lower concentrations (5 and 10 mg mL-1) and at 15, 20 and 25 mg mL-1 concentrations hemolysis was found in chick blood. Though it produced hemolysis in chick blood, no hemolysis was observed in sheep blood. However feeding experiments with other animal models are needed before using them in food intended for human consumption or to incorporate them in animal feeds.
Pyocyanin pigment from P. aeruginosa produced good colouration when mixed with agar. It showed a light blue colour which did not deterioated on boiling. The pigment did not make the agar to loose its property. The pigment seems to have a binding property with agar. Pyorubrin also had all these properties except that it gave chocolate brown colour on boiling. Many microbial pigments have already been commercialized: Astaxanthin from Xanthophyllomyces dendrorhous, pink red from Penicillium oxalicum, riboflavin from Asbhya gossypii. Like wise P. aeruginosa pigments also has the potential to use as colourant in beverages, cakes, confectionaries, puddings etc. the pigments may also be used to decorate and display the food items also.
Thus the results of the present study clearly indicated that the phenazine pigments, pyocyanin and pyorubrin of P. aeruginosa can be produced in laboratory. The results of antibacterial and hemolytic activity were also favourable. The colour obtained with agar also pleasant to see. Hence, the pigments seem to have the potential to use as food colourants. However, further research is needed before using these pigments as food colourants in human food.