Media, Chemicals and Instrumentation Used
Lemon pickle was obtained locally (Amritapuri, Kollam, Kerala, India) and rice water was aseptically fermented for 6 hours. De Man, Rogosa and Sharpe agar (MRS agar), and Nutrient Gelatin was obtained from HiMedia, India for isolation of Lactobacillus strains and gelatinase activity respectively. Gelatin was obtained from HiMedia, India and Heparin from Biological E. Limited, Hyderabad, India for matrix binding studies. Chromocult® Coliform Agar was purchased from Merck, India. Bile salts, Pepsin and Ortho-nitro phenyl –D-galactopyranoside discs (ONPG) discs were procured from HiMedia, India. Solvents, hexadecane and xylene was procured from Sigma-Aldrich, India. Dialysis tubing (3.5K MWCO) was purchased from Thermo Fischer Scientific, India. Absorbance measurements employed synergy microplate reader using Gen5.2.05 Software. Fluorescent microscope (Olympus IX71) and (Olympus BX51) was employed for matrix binding studies.
Microbial strains used in the study
Bacillus clausii spore suspensions (Enterogermina®), Klebsiella pneumoniae (MTCC 3384) from Microbial Type Culture Collection, Chandigarh, India, Pseudomonas aeruginosa wild type strain, PAO1 (ATCC 15692), from American Type Culture Collection, Manassas, VA, USA , Escherichia coli ET (enterotoxigenic), Escherichia coli (MDR), Salmonella enterica and Staphylococcus aureus was gifted by Dr. Bhabatosh Das, THSTI, Faridabad, India.
Isolation of probiotic strains from rice water and lemon pickle
Locally available lemon pickle and fermented rice water were chosen to isolate the Lactobacillus strains in MRS agar at 37 °C by spread plate method. The rice water sampled from cooked rice was aseptically fermented for 6 h and serially diluted up to 105. In both cases, few randomly selected colonies were further screened on its binding to gelatin. Gelatin coating was performed in flat bottom 96 well plates by incubating 100 µL of 10 mg/mL gelatin in each well at room temperature for 1 h followed by rinsing with phosphate buffer saline (PBS) thrice. The MRS positive colonies suspended in PBS were added to each well and kept at room temperature. After 10min, the bacterial suspension was discarded and washed with 100 µL PBS 5 times and the fifth wash was spread plated. More stringent similar volume wash was done subsequently with 1 N NaCl in PBS and then 1% dimethyl sulphoxide (DMSO) in PBS. The uniform small pinheaded colonies obtained from 1% DMSO-PBS fifth wash, which is considered as a strong binder to the gelatin, was selected and subcultured for characterization. Rice water isolate was termed RS and pickle isolate as T1 (Kumar and Ghosh 2012).
Genomic DNA isolation and 16SrRNA sequencing for identification of strains
Genomic DNA isolation was performed by phenol chloroform method (Green and Sambrook 2017; Xu et al. 2019; Porayath et al. 2018). The DNA isolated was subjected to normal PCR with the help of 16s rRNA gene primers forward primer (5’-AGAGTTTGATCCTGGCTCAG–3’), reverse primer (5’-ACGGCTACCTTGTTACGACTT–3’) producing an amplicon of length 1.5 kb. After sequencing BLAST analysis was performed (Zhang et al. 2000; Salim et al. 2019).
BLASTn analysis of the sequences were done with the default parameters. The results obtained for aligned fasta sequences were downloaded from the NCBI BLAST and phylogenetic analysis was carried out with the help of MEGA-X version 10.0.5. Fasta files were imported and aligned using MUSCLE and the resulting.meg files were used to construct the phylogenetic tree using the Neighbor-Joining method with default parameters (Saitou and Nei 1987;.Tamura, Nei and Kumar 2004; Kumar et al. 2018).
Assessment of probiotic properties of the strains
Acid tolerance ability
To determine the acid tolerance of the strains, overnight culture of the strains (were inoculated into tubes of MRS broth previously adjusted to pH values (1.5 and 3) using 1N HCl and 1N NaOH. The cultures adjusted to 0.1 OD (in accordance with McFarland Standard) was inoculated and aliquots of cultures exposed to pH 1.5 and 3 at 0 h and 3 h were plated on to MRS agar and viable counts were determined. MRS broth maintained at pH 7 was used as the control (Kim et al. 2019). Experiments were repeated in triplicates and average of 3 independent values were plotted.
Bile Tolerance ability
The bile salt tolerance was checked in MRS agar incorporated with bile salts, adding varying concentrations from 0.2% to 2%. Aliquots of overnight cultures was spread plated onto the surface of the bile-salt-containing MRS agar at 0.2% and 0.5% and viable counts were taken after 3 h of exposure. MRS broth without bile salts was used as control (González-Vázquez et al. 2015).
Tolerance to Simulated Gastric Juice (SGJ):
The strains were centrifuged and resuspended in saline equivalent to an absorbance value of 1 at 600 nm. The cultures were then inoculated to simulated gastric juice (125 mM NaCl, 17 mM KCl, 45 mM NaHCO3, 3 g Pepsin) adjusted to 3 different pH ranges 2, 3 and 7. Absorbance values after exposure to 6 h was measured at 600 nm (Hassanzadazar et al. 2012).
Elucidation of cell surface properties
Microbial Adhesion to Hydrocarbon Test (MATH) assay for hydrophobicity
Bacterial cell surface hydrophobicity was assessed by measuring adhesion to hydrocarbons, hexadecane and xylene. Overnight cultures of RS and T1 were centrifuged at 9,000 g for 10 min at 4 ℃. The pellet was washed with phosphate urea magnesium buffer (PUM buffer- 22.3 g K2HPO4, 7.26 g KH2PO4, 1.80 g urea, 0.2 g MgSO4.H2O, pH 7.1). Absorbance at 600 nm was adjusted to 1 OD. To 5 mL cell suspension 1 mL hexadecane was added. The two-phase system was vortexed for 2 min followed by incubation at 37 ℃ for 1 h. Suspension was vortexed for 3 min and then incubated at room temperature for 1 h. Aqueous phase was removed, and absorbance was measured at 600 nm. Similarly, a two-phase system with xylene was performed. The percentage of cell surface hydrophobicity was expressed as (H %) = (1-A1 /A0) ×100 where A1 represents the absorbance of aqueous phase after 1 h and A0, at time t = 0. Bacillus clausii, a known probiotic organism was used as the positive control (Xu et al. 2019).
The strains (RS and T1) were grown in MRS broth and incubated at 37 ℃. The overnight cultures were centrifuged (5,000 g, 15 mins, 4 ℃), harvested cells washed twice with phosphate buffer saline (PBS) and resuspended in the same to 4 mL calibrated to an OD of 0.2 (108 CFU/mL). The cells were vortexed for 10 s and the autoaggregation was determined at 3, 5 and 24 h respectively. The top portion of the suspension was transferred to another tube with 3.9 mL of PBS and the absorbance (A600) was measured each hour. The autoaggregation percentage is expressed as: Autoaggregation (%) = [1—At/A0] × 100 Where At denotes the absorbance at time t = 1- 5 h and 24 h and A0 the absorbance at t = 0. Bacillus clausii, a known probiotic organism was used as the positive control (Sorroche et al. 2012; Ben Taheur et al. 2016).
Coaggregation assay of probiotic strains with pathogenic strains
Coaggregation of probiotic strains with pathogens was investigated. The bacterial cell suspension was resuspended in PBS to approximately 108 CFU/mL, and 2 mL of each bacterial suspension was mixed with 2 mL of RS and T1 each and vortexed for 10 s. (Salmonella enterica, E. coli MDR, Klebsiella pneumoniae).. Control tubes, each with 4 mL of bacterial suspension alone, were maintained at room temperature. The degree of coaggregation was measured at 600 nm for 3 h, 5 h and 24 h respectively (Collado, Meriluoto and Salminen 2008).
Bacterial attachment to immobilized extracellular matrix (ECM) proteins
Gelatin and heparin (1 mg/mL, 50 μL) was added to 96 well plates and was incubated at room temperature for 1 h. Unbound proteins were removed by washing with PBS. Absorbance of the strains was adjusted to 0.1 (600 nm) and 50 μL of each of the bacterial suspension was transferred to the coated plates and incubated for 1 h at room temperature. The wells were washed with 1X PBS for 3 times, 50 μL of formalin was added and the plate was kept undisturbed for 20 min to fix the bacteria and was washed with PBS. Crystal violet (0.1 %, 50 μL) stain was added for 1 minute. Excess stain was removed by washing and the plates were dried after which 50 μL of acetic acid was added into the wells to solubilize the dye. Absorbance was measured at 595 nm in Synergy microplate reader using Gen5.2.05 Software. Bacillus clausii and E. coli ET strain were used as controls (Yadav et al. 2014; Nishiyama et al. 2015).
Microscopic examination of binding efficiency of the strains (fluorescent staining and crystal violet staining)
Microscopic observation of the probiotic binding to ECM was performed with some modifications (Shahara et al. 2012). Cultures (RS and T1 in MRS broth) adjusted to 0.1 OD were inoculated into sterile coverslips placed in 6 well micro titre plates and were kept for incubation for 5 h. The wells were washed with 1 X PBS to remove planktonic cells and coverslips transferred to glass slides were heat fixed at 55 ℃ for 20min. Slides were then kept overnight at 4 ℃ and air dried (15 min). Fixed slides were stained with 0.02% acridine orange for threemin in the dark, washed with distilled water and then air dried at room temperature for 15 min. The attached cells were then imaged using a fluorescent microscope (Olympus IX71). Another set of matrix bound coverslips were stained with crystal violet (0.1%) and observed under 100 X magnification (Olympus BX51).
Biofilm formation of probiotic strains
In order to perform biofilm quantification, 2 mL of overnight culture of RS and T1 were inoculated into 6 well microtitre plates, with an initial turbidity of 0.25 OD at 600 nm. MRS broth was employed as control. Plates were then left in a static condition for 48 h at 30 °C. Biofilm formation was analysed by standard crystal violet assay as described by Gómez et al. Quantification was made based on the equations, non-biofilm producers [OD ≤ ODC], weak biofilm producers [ODC < OD ≤ 2 x ODC], moderate biofilm producers [2 x ODC <OD ≤ 4x ODC], strong biofilm producers [4 x ODC < OD] where ODC (cut-off) is mean OD value of control. B. clausii was kept as a positive control. Visualization of biofilm formation was further performed using acridine orange and crystal violet staining (Gómez et al.2016).
Detection of β -galactosidase activity:
Qualitative determination of the rate of lactose fermentation was done by β-galactosidase assay. A single colony of RS and T1 was applied to Ortho-nitro phenyl β –D-galactopyranoside discs (ONPG) in MIC tubes followed by addition of 100 μL of saline. Reduction of ONPG to ONP (ortho nitrophenol) was indicated by a color change to yellow at room temperature (Gómez et al 2016; Cebeci and Gürakan 2003).
Coliform reduction in sewage aided with probiotic biofilters
The efficiency of the probiotic strains, T1 and Bacillus clausii for their ability to form biofilms embedded in biofilters was utilized to reduce coliform count in sewage. Pseudomonas aeruginosa (PAO1)was used as a positive control. 1 L bottle employed as filter was filled with pre washed and dried sand, charcoal, coarse gravel, fine gravel, big gravel in the ratio of 3:3:1:1:1 respectively to a total bed height of 18 cm.The filter was aseptically washed for 30 min, followed by inoculation of overnight culture of T1, Bacillus clausii and Pseudomonas aeruginosa at OD (600) of 0.3 into the respective filters for 72 h. A control filter was maintained devoid of biofilm. After 3 d, the biofilter was washed with 0.9% percent saline without disturbing the biofilm and sewage was introduced at a flow rate of 35 mL/min (slow sand filter linear velocity: 0.3 m/h). The reduction in fecal coliform (Escherichia coli) was checked in Chromocult® Coliform Agar (Verschuere et al. 2000; Prol-García and Pintado 2013).
Detection of Gelatinase activity
Gelatinase production of the strains was checked by spotting 1µL aliquots of the 24 h cultures on to the surface of nutrient gelatin plates (HIMEDIA). Plates were incubated for different time period and temperature, 37 oC, 42 oC (48 h), 25 oC (72 h) and 10 oC and 15 oC (10 d). After incubation, the plates were maintained at 4 oC for 2 h and gelatin hydrolysis was recorded as opaque halos around the colonies (Oh et al 2018).
Bacteriocin extraction by pH mediated adsorption desorption method and activity assay:
Initially, heating the culture broth to 60 ℃ was done in order to prevent the inactivation of bacteriocin by proteases present in the culture medium. Adsorption of bacteriocin to producer cells was facilitated by adjusting the pH to 6.0 using 1M NaOH followed by steering it for 30 min at 4 ℃. The cells were harvested by centrifuging at 10,000 g for 25 min at 4 °C and pellets were washed twice with sterile 0.1 M phosphate buffer (pH 6.5). Pellets resuspended in 100 mM NaCl, was adjusted to pH 2.0 (1N HCl), stirred for 12 h at 4 ℃. Centrifugation of cell suspension at 10,000 g for 25 min was done and supernatant dialyzed against distilled water at 4 ℃ for 24 h (dialysis tubing 3.5 K MWCO, Thermo Fischer Scientific). The protein concentration was determined by Bradford assay (Elegado, Kim and Kwon 1997; Zhang et al. 2009; Zhang et al. 2013).The dialyzed samples were tested against indicator pathogens S. typhi, S. aureus and K. pnuemoniae by microtitre inhibition assay (Vijayakumar and Muriana 2015).
Statistical analysis of data obtained was performed by conducting Two-way RM ANOVA and values were expressed as mean ± SD (Standard deviation of the mean) values of 3 independent experiments using the software Graph Pad Prism 6. Significance levels were at *P ≤ 0.05,**P ≤ 0.01,***P ≤ 0.001 and ****P ≤ 0.0001.