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Richlin Machado T et al.

, IJSID, 2013, 3 (4), 478-483



International Journal of Science Innovations and Discoveries

Research Article

An International peer Review Journal for Science

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ANTI ADHESIVE, ANTIMICROBIAL AND BIODEGRADABILITY ASSAY OF A LIPOPEPTIDE BIOSURFACTANT FROM Richlin Machado. T1, Ajaz haja mohideen. R2, Saravanakumari, M3 and Prabhavathi. P4* Tirunelveli; 3GRD College, Coimbatore, Tamil Nadu, India; 4PSG College of Arts and Science, Coimbatore, Tamil Nadu, India.

of Microbiology, St. Maries College, Tuticorin; 2Department of Microbiology, Sadakathullah Appa College,

Received: 10-07-2013 Accepted: 14-08-2013

*Corresponding Author

ABSTRACT Biosurfactants are valuable microbial amphiphilic molecules with effective surface active

and biological properties applicable to several industries and their processes. Microbes synthesize them, especially during growth on water immiscible substrates, providing an ecological acceptability. These molecules could be widely used in cosmetic, alternative chemically prepared conventional surfactants. In recent years natural pharmaceutical and food processes as emulsifiers, preservatives and detergents and in biosurfactants have attracted attention because of their low toxicity, biodegradability and bioremediation processes. The present study was aimed to screen and to evaluate the Lactococcus lactis was isolated from homemade curd was found to be a potent producer of the present work intended to purify the biosurfactant produced by the indigenous purified bioremediation and crude oil degradation. biosurfactant in microbial decolonization, bacteriocidal, heavy lipopeptide biosurfactant with olive oil as carbon source. In continuation of these studies, probiotic bacteria L. lactis, structural analysis of the biosurfactant and to apply the Keywords: biosurfactant, metal resistant bacteria, TLC. metal

Address: Name: Prabhavathi. P Place: Tamil Nadu, India E-mail:

biosurfactant and heavy metal resistant activity of biosurfactant produced by L. lactis.

International Journal of Science Innovations and Discoveries, Volume 3, Issue 4, July-August 2013


Richlin Machado T et al., IJSID, 2013, 3 (4), 478-483 INTRODUCTION are excreted extracellularly in the growth medium. In recent years, interest in biosurfactants increased due to their possible applications in environmental protection, crude oil drilling and in the pharmaceutical and food processing industries (Bodour et al., 2004). There are different types of bacteria and unicellular yeasts produces biosurfactant in natural environment. erythropolis, Arthrobacter sp. They produce biosurfactants from substrates such as pyruvate, citrate, glycerol, olive oil and Among the most studied bacteria are Pseudomonas aeruginosa, Micrococcus sp., Bacillus sp., Lactococcus lactis, Rhodococcus fructose. Many studies have also proven that the biodegradation time for microbes can be shortened in the presence of biosurfactants (Harshey and Matsuyama, 1954). Probiotic bacteria are friendly to human health. One of the most important oxidative metabolism. It is extensively used in the manufacture of dairy products and production of antimicrobials that are biosurfactant producing probiotic bacteria is Lactococcus lactis. L. lactis is aerobic, chemo organotropic organisms with used as natural food preservatives. L. lactis is now being used in the area of medicine, as a vaccine delivery vehicle to the gastrointestinal tract under nutrient limiting situation of L. lactis. To date, interest has focused principally on the use of and synthetic surfactants can enhance either the chemical removal or the biodegradative removal of inorganic contaminants surfactants to remove inorganic contaminants from soil. Studies of inorganic contaminants have shown that both biological from soil. While these studies indicate the potential for use of surfactants to facilitate the removal of inorganic materials from soil, the literature contains very little actual information concerning surfactant removal of metal contaminants. The present lactis. In continuation of these studies, the present work intended to purify the biosurfactant produced by the indigenous decolonization, bacteriocidal, heavy metal bioremediation and crude oil degradation. MATERIALS AND METHODS Isolation and identification of Lactococcus Lactis curd sample was aseptically transferred into a 250 ml flask containing sterile 100 ml of mineral salt medium with 2% paraffin as the carbon source and was incubated at 28 C for 48 hours (De Man et al., 1960). CONFIRMATION OF BIOSURFACTANT ACTIVITY Haemolytic activity 48 hours. The isolated organism was tested for haemolytic activity by plating the cells in blood agar and incubated at 37C for The isolate was grown for 48 hours at room temperature in minimal media. The cultures were centrifuged at Homemade curd sample, fermented product of milk was collected in a sterile container aseptically. Collected 10ml of the probiotic bacteria L. lactis, structural analysis of the biosurfactant and to apply the purified biosurfactant in microbial study was aimed to screen and to evaluate the biosurfactant and heavy metal resistant activity of biosurfactant produced by L. Biosurfactant are amphiphilic compounds produced by the microorganisms, which either adhere to cell surfaces or

Emulsification activity and index 5000rpm for 20 minutes and the cell free supernatants were used for the test. Two sets of 2 ml of the cell free broth was mixed with 2 ml of paraffin oil and vortexed for a minute and left undisturbed. After one hour the optical density of oil in water emulsion phase was measured at 610 nm for one set. The second set was left undisturbed for 24 hours. The height of emulsified layer was measured and E24 was calculated (AbuRuwaida, et al., 1991). E24 = Total height of liquid column Height of emulsified layer (cm) 100

International Journal of Science Innovations and Discoveries, Volume 3, Issue 4, July-August 2013


Richlin Machado T et al., IJSID, 2013, 3 (4), 478-483 Analysis of biosurfactant by TLC for lipid and amino acid different standard amino acids and adipic acid. The spots were developed with solvent system boric acid: acetic acid: water system isopropanol: water: 25% ammonium hydroxide (80:11:9). Developed spots were detected using iodine vapours. Effect of various parameters on biosurfactant activity Hi media TLC plates were spotted with purified biosurfactant at 10 mg/ml concentration in methanol along with

(75:10:15) for amino acid. Developed spots were detected using Ninhydrin in acetone. The spots were developed with solvent To determine the thermal stability of the produced biosurfactant 2ml of the cell free broth was maintained at different

temperatures of 20C , 30C , 40C , 50C , 60C , 70C , 80C and 90C for 15 min and cooled to room temperature. To

determine the effect of pH on activity, the pH of the culture supernatant was adjusted to 2, 3, 4, 5, 6, 7, 8, 9 and 10. The effect of addition of different concentration of NaCl on the activity of biosurfactant was determined similarly. The biosurfactant was (E24) was determined (Makkar, 1998). Agar well diffusion test prepared by mixing 1 ml of inoculum with 15 ml respective medium (LB) at 45C and allowed to set and wells of 8 mm incubated at 37C during 24hrs for bacterial strains (Reid et al., 2001). Anti-adhesive assay The well diffusion assay was used to evaluate the antimicrobial activity of biosurfactant, the microorganisms were ASSAY OF ANTIMICROBIAL ACTIVITY OF BIOSURFACTANT redissolved in distilled water at specific concentration of NaCl of 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0 and 4.5. The emulsification index

diameter were made. About 30l of 10mg / ml concentration of the purified biosurfactant was added to each well and Each well of a sterile 96-well micro titer plate was filled with 200 l biosurfactant of dif ferent concentrations

(ranging from 2.5 to 4.0 g/L) and incubated for 24 h at 4C, and then the plates were washed twice with Phosphate buffered

saline (PBS). Control wells contained PBS only (Velraeds et al., 1996), about 200l aliquote of pathogenic microbial suspension positive control was maintained by adding 200l of microbial suspension without biosurfactant. Unattached organisms were removed by washing the wells three times with PBS. The adherent microorganisms were fixed with 200l of 99% methanol at room temperature for 5 min. Excess stain was rinsed off by placing the plate under running tap water. Subsequently the acid per well. The absorbance of the dissolved dye, corresponding to the number of adherent microorganisms, was measured in a microplate reader at 595nm. The percentage reduction in adherence was calculated using the following equation: BIODEGRADATION OF CRUDE OIL Oil spreading assay (Rodrigues et al., 2006) On the thin layer, 10l of biosurfactant (10mg / ml concentration) was gently placed at the center. Clearance of zone was observed. About 10l of crude oil was placed over the surface of 40ml of water in a petriplate which forms a thin layer. % Reduction in adherence = [(A control) (A sample)] /A control100]

of Pseudomonas sp, Enterococcus sp, Salmonella sp, and Candida sp were added and incubated in the wells for 4 h at 4C. A per well, and after 10 min the wells were emptied and left to dry. Then the wells were stained with 1% crystal violet solution plates were air dried, the dye bound to the adherent microorganisms was resolubilized with 200l of 33% (v/v) glacial acetic

International Journal of Science Innovations and Discoveries, Volume 3, Issue 4, July-August 2013


Richlin Machado T et al., IJSID, 2013, 3 (4), 478-483 RESULTS AND DISCUSSION Identification of isolate was identified by specific primer as Lactococcus lactis. Maximum identity of 90% was identified. Quantification of biosurfactant production The microscopic and biochemical techniques were performed for the isolate. The 16S rRNA isolated from the isolate Biosurfactant activity was measured by reduction in surface tension of the medium and emulsion formation by

measuring emulsification index and emulsification activity as 72% and 0.26 nm. Pure white, powdery 0.1gm of biosurfactant from 100ml of 48 hrs old culture supernatant was purified. Qualitative analysis of biosurfactant by TLC After exposure to iodine vapors, the Rf value was determined as 0.81. So the presumed lipid moiety can be lipid. After

exposure to ninhydrin, Rf value was determined as 0.54, 0.34 and 0.11. So the presumed amino acid moiety can be Methionine, Alanine and Lysine. Effect of various parameters on biosurfactant activity and the results were tabulated pH EI TEMP EI 61.1 10 C 92.5 2 Biosurfactant suspension subjected to various pH, Temperature and NaCl was calculated for emulsification index (EI) 3 4 5 6 7 69.9 60 C 55.5 2.5 59.2 3 8 9 10

Table 1: Determination of EI at varying pH, temperature and salt concentrations 61.1 20 C 93.5 0.5 61.1 30 C 92.8 1 62.5 40 C 72 1.5 75 2 67.8 53.6 50 C 56.5 64.4 70 C 76.9 3.5 61.1 80 C 83.3 4 66.5 90 C 92.5 4.5

NaCl (g/100ml) EI

production was indirectly measured using its activities such as surfactant and emulsification properties. L. lactis reduced the surface tension of the medium from 0.072 N/m to 0.052 N/m and emulsified the paraffin in water to 72% with a foam stability isolate. It has formed stable foam for 10 minutes. Because of the stable emulsion formation ability of the isolate and (Muthusamy et al., 2008). ANTIMICROBIAL ACTIVITY Agar well diffusion method and Enterococcus sp. The biosurfactant showed antibacterial activity against Pseudomonas sp. The diameter of the zone was measured about 4.8cms. International Journal of Science Innovations and Discoveries, Volume 3, Issue 4, July-August 2013 The purified biosurfactant was used for antibacterial activity against Pseudomonas sp, Salmonella sp, Escherichia coli of 10 minutes. Surfactant activity of commercially used biosurfactant producers such as Pseudomonas sp., Candida sp., and Bacillus sp., are 0. 036 0.040 N/m (Velraeds et al., 1996; Rodrigues et al., 2000) which is comparatively very low in the emulsification properties as similar to commercial strains, it can be exploited in industries for degreasing and food processing

The L. lactis organism was inoculated in mineral salt medium with paraffin as carbon source and biosurfactant








Richlin Machado T et al., IJSID, 2013, 3 (4), 478-483 Anti- adhesive assay significantly inhibited the adhesion of Candida sp, Pseudomonas sp, Salmonella sp, E. coli and Enterococcus sp. Anti-adhesive assay of biosurfactant against clinical pathogens Con ( gm / l ) 2.5 3.0 3.5 4.0 4.5 Oil spreading assay measured about 2.6cms which indicated the biosurfactant has the ability to degrade crude oil. Enhanced biodegradation of crude oil The extracted surface active compound showed zone of clearance on crude oil. The diameter of the zone was On 20th day of observation color changes occurred on all the plates. This clearly indicates that the biosurfactant has Candida sp 5.5% 14.8% 29.6% 44.4% 9.2% Pseudomonas sp 29.7% 45.8% 46.2% 48.7% 49.5% E. coli 16.9% 20.3% 30.5% 45.7% 54.2% Enterococcus sp 10.2% 16.1% 19.1% 36.7% 44.1% The anti-adhesive activity of the biosurfactant against clinical pathogens was screened. The biosurfactant was found Salmonella sp 11.4% 12.8% 31.4% 51.4% 20%


enhanced biodegradation of the crude oil. Crude oil degradation by purified biosurfactant and isolate in sand mixed with crude oil along with added nitrogen sources were evaluated. Crude oil was efficiently degraded (turned from black to light brown color) by 10th day in inoculum without addition of nitrogen source, wherein other cases it took 20 days for degradation (color reduction). Moreover the crude oil mixture inoculated with isolate immediately degraded than the applied pure biosurfactant. human/animal health (Rodrigues et al., 2006). CONCLUSION So, L. lactis can also be used for the bioremediation of crude oil, as it is a probiotic it may cause any alarming effect on The curd is obtained by fermentation of milk by the probiotic bacteria, there are several bacteria which produce

different types of enzymes, vitamins and minerals in curd that complement human health. Certain bacteria produces the biosurfactant in curd thus try to eliminate the pathogenic bacteria from curd by antagonistic activity. So for the isolation probiotic bacteria curd sample was opted for the study. 1. 2. 3. 4. 5. REFERENCES biosurfactant producing bacteria product AbuRuwaida AS, Banat M, Haditirto, Salem S, Kadri A (1991). Isolation of characterization and evaluation. Acta Biotech. 11(4): 315-24. Lactobacillus sp. Appl. Environ. Microbiol. 59: 34-39.

Bodour PL, Blomberg L, Hendriksson A (2004). Inhibition of adhesion of Escherichia coli K88 to piglet ileal mucus by De Man JC, Rogosa M, Sharpe ME (1960). A medium for cultivation of Lactobacilli. J.Apllied Bacteriol, 23: 130-135. Pseudomonas aeruginosa Proc. Natl. Acad. Sci., 91: 8631-8635. Bacillus subtilis, Journal of industrial microbiology, 20: 48-52. Harshey RM, Matsuyama T (1954). The enzymatic synthesis of a rhamnose-containing glycolipid by extracts of Makkar RS, Cameotra S (1998). Production of biosurfactant at mesophilic and thermophilic condition by a strain of International Journal of Science Innovations and Discoveries, Volume 3, Issue 4, July-August 2013


Richlin Machado T et al., IJSID, 2013, 3 (4), 478-483 6. 7. 8. 9. Muthusamy Datta KKR, Srinivasan B, Balaram H, Eswaramoorthy (2008). Synthesis of agarose-metal/semiconductor 120 (6): 579-586. 555-62.

nanoparticles having superior bacteriocidal activity and their simple conversion to metal-carbon composites. J. Chem. Sci, Reid G, Bruce A, Smeianov V (1998). The role of Lactobacilli in preventing urogenital and intestine infections. Int Dairy 8: Rodrigues L, Banat IM, Teixeira J, Oliveira R (2000). Biosurfactants: Potential applications in medicine. J. Antimicrobial Chemotherapy 57: 609-618. Rodrigues L, Moldes M, Teixeira J, Oliveira R (2006). Kinetic study of fermentative biosurfactant production by Lactobacillus strains. Biochem. Eng. J., 28: 109-116. faecalis by biosurfactants from Lactobacillus isolates. Appl Environ Microbiol; 62(6):1958-63.

10. Velraeds MM, Van der Mei HC, Reid G, Busscher HJ (1996) . Inhibition of initial adhesion of uropathogenic Enterococcus

International Journal of Science Innovations and Discoveries, Volume 3, Issue 4, July-August 2013