Isolation and Characterization of Lactobacillus
Lactobacillus was isolated by serial dilution technique from home-cured curd samples and was plated onto pre sterilized MRS (Man, Rogosa and Sharpe) agar plates. Then the plates were incubated at 37°C for 24 h. Then the colonies with completely different morphology, color and dimension were selected and sub-cultured on recent MRS agar plates. The organism for biofilm production was obtained from domestic sewage sample. The human infective gram negative bacterium Escherichia coli was isolated and confirmed by culturing on (EMB Agar) Eosin methylene blue agar.

The selected colonies were identified supported morphological and biochemical characteristics. The morphological characteristics like colony size, surface, opacity, color, motility and cell shape were discovered and gram staining was performed. Biochemical characteristics like Indole production, methyl Red, Vogues Proskauer, Citrate, Catalase, Oxidase, Starch reaction and Casein degradation were performed (Holt et al., 1994).

Amplification and sequencing of 16S rRNA sequence analysis:
Genomic deoxyribonucleic acid was extracted as per Ausubel et al., (1988). 16S rRNA genes of the microorganism isolates were amplified with genomic deoxyribonucleic acid isolates as sequence templates and 8F and 1490R primers every reaction mixture contained 2 µl of template deoxyribonucleic acid (100 ng), 0.5 µM of 2 primers, and 25 µl of accelerator Master combine (Bioron). The PCR program consisted of associate initial denaturation step at 94°C for five min, followed by thirty cycles of deoxyribonucleic acid denaturation at 92°C for thirty sec, primer hardening at 50°C for one min, and primer extension at 72°C for two min was administered in Thermal Cycler (ThermoHybaid). The PCR end products were refined by QIAquick PCR purification kit and cloned with QIAGEN PCR biological research kit. Clones were identified and isolated, plasmids with insert were sequenced with M13 Sequencing Primers in ABI Biosystems automatic sequencer. Nucleotide database was searched with the sequences obtained with NVBI BLAST (Blastn) tool (http://www/ncbi.nlm.nih.gov/BLAST) (Altschul et al., 1997).

Biosurfactant Production
A starter culture of 20 ml MRS broth was prepared with Lactobacillus and incubated for 24 h at 28°C in static condition. After 24 h of incubation the 20 ml broth starter culture was inoculated into 1000 ml broth and incubated at 28°C for 24 h. After incubation, the cells were removed by centrifugation at 8000 rpm for 30 minutes. The supernatant was collected.

Screening tests for Biosurfactant
1ml of oil (motor oil) was added on the surface 20 ml of distilled water in a petriplate, and 0.5 ml of supernatant was added to the surface of oil.  Formation of a clear zone is the indication of biosurfactant presence in the supernatant. The diameter of the zone was measured.

Determination of emulsification index
2 ml sample of the cell free supernatant was taken in a small test tube along with 2 ml of oil (petrol). To ensure homogenous mixing of both the lipids, the mixture was vortexed at high speed for 2 minutes and incubated at RT for 24 h. The emulsification index (E₂₄) was calculated by dividing the measured height of the emulsification layer by the mixtures total height and multiplying by 100. (Anayata Sharma et al., 2014).

Drop collapse test
25µl of biosurfactant was pipetted as a droplet onto a parafilm, the spreading of the droplet on the parafilm was observed. Similarly 25µl of water was pipetted on a parafilm and the changes were observed. The biosurfactant spreads and get flattened whereas water stays intact without any change. (Arezoo Tabmourespour et al., 2011).

Isolation, Extraction and Purification of Biosurfactant
The supernatant after centrifugation was collected and the pH was adjusted to 2.0 using 6 N HCL and kept at 4°C overnight. Equal amount of Chloroform: Methanol (2:1) mixture was added to the supernatant, vigorously mixed and kept overnight for phase separation. The mixture forms 3 layers – lower layer is the chloroform: methanol, middle layer is the biosurfactant and upper layer is the supernatant (Abdullahi Adamu et al., 2015). The biosurfactant formed (middle layer) was carefully taken out with the help of a micropipette and was transferred to microfuge tubes. 1 ml of distilled water was added to the microfuge tube containing biosurfactant and was thoroughly vortexed. This mixture was centrifuged at 7000 rpm for 30 minutes at 4 C. The supernatant was discarded and the pellet obtained was allowed to dry for 24 h.

Analysis and Identification of Biosurfactant

Thin Layer Chromatography (TLC)
The identification of the bioactive compound present in the biosurfactant was screened by Thin Layer Chromatography (TLC). Silica coated plates were prepared and the pelleted crude biosurfactant obtained was spotted onto the plate. The solvent used for separation was chloroform: water: methanol (65:24:4). Ninhydrin reagent was sprayed over it and the appearance of red spots indicates the presence of lipopeptide in the biosurfactant. (Anayata Sharma et al., 2014).

FTIR analysis of biosurfactant
To demonstrate the functional groups of the bioactive compound within the biosurfactant, the pellet was analyzed by Fourier transform infrared spectroscopy (FTIR) (Ragavendran et al., 2011). The spectrum for knowing the functional groups dominating within the samples was done by using model Perkin Elmer Spectrum Version 10.03.07 (Courtesy: Bangalore Testing laboratory Pvt. Ltd.) NDXUS-672. The spectrum was taken in a very mixed IR 400 -4000 cm^-2 with 16 scan speed and was recorded in an Attended total reflectometer (ATR)

Antimicrobial Activity of Biosurfactant
A total of 9 plates were used and all the plates were swabbed with Escherichia coli on MHA (Muller Hinton Agar) plates. In four plates, two wells were made and labelled as 1 and 2 respectively. To the well labelled 1, purified biosurfactant was added and to the well labelled 2, different concentrations of SDS (sodium dodecyl sulphate) – 0.1%, 0.5%, 1%, 1.5% were added in order to compare the action of biosurfactant against chemical surfactant. To the remaining 5 plates, one well was made for each them, for which three plates were crude biosurfactant, for the last two plates, one was supernatant and the other was MRS broth which served as negative control. A clear zone around the wells indicates the antimicrobial activity of biosurfactant (Anayata Sharma et al., 2014). The results were tabulated and analyzed.

Inhibition Study on Biofilm Formation
Air Liquid Interphase Coverslip Assay
E.coli was cultivated with LB medium in six Erlynmeyer flasks (50 ml). Sterile cover slips were added to the broth culture and incubated at 37°C for 24 h. After incubation, the cover glass was taken out from one flask which serves as the control and stained with crystal violet for 1 minute and observed under microscope at 40x magnification to check for the presence of biofilm. Later the coverslips were treated with biosurfactant at different concentrations (10 %, 20%, 30%, 40%, 50% v/v) for 6 h at room temperature. After incubation, the cover glass was taken out and washed with distilled water and stained with crystal violet for 1 minute. Under 40 X objective the action of biosurfactant on the bifolm was bserved using a microscope (Pratt et al., 1999).

Biofilm Inhibition Assay
Overnight culture of E.coli was inoculated in to six microfuge tube containing 0.5 ml of LB broth and 0.5ml of different concentrations of biosurfactant (10%, 20%, 30%, 40%, 50% v/v) were added to 5 microfuge tubes and 1 microfuge tube served as a control without biosurfactant. The tubes were incubated for 48 h at 37°C, later 0.1 ml crystal violet was added to all the microfuge tubes and exposed for 5 minutes.  Following which the stain was discarded and the microfuge tubes were rinsed twice with distilled water. Then the microfuge tubes were allowed to dry and the activity of biosurfactant was observed based on the colour intensity of crystal violet on the microfuge tube.

RESULTS AND DISCUSSION

The biosurfactant producing Lactobacillus strain was isolated from homemade curd sample and confirmed by its morphological and biochemical characteristics (Table 1), (Table 2). After gram staining, the biochemical tests were performed. The DNA was isolated and its molecular characterization was done using DNA sequencing technique. The strain was used for isolation and extraction of biosurfactant (Table 3). The isolated organism was subjected for different screening techniques to check for the production of biosurfactant.  The biosurfactant extracted from Lactobacillus displaced oil and formed a clear zone in the center of the plate and the diameter of the clear zone formed was measured as 2 cm (Fig. 1).

Table 1. Biochemical characteristics of the isolate

Table 2. Morphological characteristics of the isolate

Table 3. Molecular Identification of the isolate.

Fig. 1. Oil spread technique showing zone of clearance

The emulsification index of the biosurfactant was evaluated against substrate as petrol. The emulsification index (E₂₄₎ obtained was 58.1%. The good emulsifying property is critical for biosurfactants to be promising in different environmental and industrial applications. The criteria for determining the emulsion-stabilizing capacity of an emulsifier is its ability to maintain at least 50%, after 24 h of its formation (Willumsen and Karlson, 1997). Considering this fact that the biosurfactant produced from the isolate had a good emulsifying capacity as its emulsification index value is more than 50%. The droplet size of biosurfactant measured was 1mm larger than the one produced by distilled water. Hence, the biosurfactant droplet resulted in a collapsed droplet which indicates its effect on reduction of surface tension. A mixture is formed after the addition of equal amount of chloroform: methanol (2:1) in which the middle layer formed is the biosurfactant which is separated out for further purification after overnight evaporation. The thickness of the middle layer was measured as 5mm.The layer was carefully separated out in to a microfuge tube and purified by centrifugation at 7000rpm for 10 minutes using distilled water. After centrifugation, a white colored pellet was obtained which was the crude biosurfactant. The pellet was dried and weighed. The crude biosurfactant was measured as 3.21 gram. The identification of the bioactive compound present in the biosurfactant was done by Thin Layer Chromatography. The Rf (Retardation factor) value was 0.82. The appearances of red colored spots on the TLC plate indicate the presence of lipopetide in the biosurfactant. According to the R_f value, the compound present in the biosurfactant produced was found to be tryptophan.

The graph indicates the presence of alkene, alkyne group and conjugated di-ene in the surfactant which indicates higher surface activity (Fig. 2). Alkene and alkyl benzene components of surfactant are being significantly most effective surfactants. New anionic surfactants are derived from aromatic or substituted aromatic molecules and alkenes. Peaks at 1500-1650 cm^-1 indicates the presence of C=C alkene component (Table 4). This reveals the surfactant extracted from the organism shows maximum similarity to the formulated anionic sulphonate surfactants having higher surface tension activity and good emulsification index (Tuvell et al., 1978).

Fig. 2. FTIR – spectrum of Biosurfactant

Table 4. Analysis of FTIR spectrum (Biosurfactant)

This present study was conducted to check the antimicrobial activity of the biosurfactant produced by Lactobacillus against pathogenic E.coli. The antimicrobial activity of biosurfactant produced was compared against chemical surfactant also viz., SDS (sodium dodecyl sulphate) at different concentrations (0.1, 0.5, 1, 1.5 %). The zone of inhibition was observed for Biosurfactant confirming their antimicrobial activity against E.coli. The same study was carried out E.coli using MRS broth as the negative control (Table 5). The results observed were; Negative control showed no antimicrobial activity against E.coli. The biosurfactant showed comparatively higher antimicrobial activity than the supernatant. The antimicrobial activity of SDS against E.coli was compared (Table 6) with the zone of inhibition shown by biosurfactant, which is measured as 1.1, 1.0, 1.2, 1.2 cm respectively.

Table 5. Antimicrobial activity of biosurfactant

In this graph, it was observed that crude biosurfactant showed higher antimicrobial activity against E.coli than SDS at all the different concentrations (Figs. 3 & 4). Firstly, average values of zone of inhibition of SDS at each concentration are plotted along with values of crude biosurfactant. Biofilm of E.coli was allowed to form on a coverslip and the action of biosurfactant in different concentrations on the biofilm was tested. The microscopic examination of the coverslip after 6 h of biosurfactant treatment showed that the number of E.coli cells decreased as the concentration of biosurfactant increased. The coverslip which served as control showed a uniform distribution of E.coli cells with a dense population. The coverslip which was treated with 10, 20, 30, 40, 50%v/v of biosurfactant was observed with a decrease in population of E.coli cells (Fig. 5). For tube test, the biosurfactant at different concentrations (10, 20, 30, 40, 50% v/v) was added along with the E.coli culture to grow for 48 h (Fig. 6). Two microfuge tubes were maintained as positive and negative control. The positive control was not treated with biosurfactant and the negative control tube was only subjected to staining. The reduction in the biomass formed on biofilm was noticed on the microscopic image as the concentration of the bioactive compound increased.

Fig. 3. Graph showing comparison of antimicrobial activity of biosurfactant against E. coli

Fig. 4. Comparison of antimicrobial activity of biosurfactant with SDS.

Fig. 5. Air liquid interphase coverslip assay

Table 6. Antimicrobial activity of SDS against E.coli at different concentrations.

Fig. 6.  E.coli culture treated with biosurfactant at different concentrations. (1) – 10%, (2) -20%, (3) – 30%, (4) – 40% and (5) – 50%v/v. Positive control – untreated microfuge and negative control – microfuge with crystal violet.

In the present study, Lactobacillus was isolated with biosurfactant producing ability. The identified Lactobacillus sp., was similar in ac­cordance with those earlier identified in different envi­ronments, suggested by Beasley (2004). The oil displacement test is an indirect measurement of surface activity of bio surfactants: a larger clear area is correlated with higher surface activity (Plaza et al., 2006). The drop collapse method is based on the principle that a drop of liquid containing a bio surfactant collapses and spreads over the oily surface. There is a right away relationship between the diameter of the sample and concentration of the biosurfactant and in distinction, the drop lacking biosurfactant remains beaded thanks to the hydrophobicity of the oil surface that causes aggregation of droplets. The molecular composition of the crude biosurfactant was evaluated by FTIR, and TLC that disclosed the presence of polysaccharides and lipid together (Lipopeptide). Similar peaks for functional groups were additionally allotted to the biosurfactant obtained from Lactococcus lactis. Proton NMR of Lactococcus lactis additionally showed the similar peaks for arrangement of atom (Saravanakumari and Mani, 2010). The microorganism strains that created microcolonies on the glass surface were characterised by a special level of adhesion to polymer. The link between property of the atmosphere surface and outer membrane of a selected strain in biofilm formation and surface (polyurethane, glass) are often related to hydrophobic microorganism (Krasowska & Sigler, 2014). Ninhydrin chemical agent disclosed the presence of liopeptide compound within the biosurfactant extracted from eubacterium as red spots, this can be quite contrastive to earlier reports on antimicrobial actions of the biosurfacants wherever the lipopeptide biosurfactants are reported to move principally against most of the microorganism (Singh and Cameotra, 2004). The antibiogram of the isolated strains secreting biosurfactants were shown to own numerous antibiotic resistance profiles, indicating their diversity and condition in numerous conditions.