2.1. Collection of samples and isolation of bacteria
A preliminary survey was made to identify the potential food products that are frequently consumed by the people of Cachar district of Assam, India. The study strictly relies on indigenous fermented rice product, commonly reported to have a beneficial effect during indigestion and constipation. White rice sample of “Ranjit” variety was collected, cooked and soaked overnight for 10-12 hrs in sterile water (Goswami et al. 2016). Samples were serially diluted (10-1-10-3 fold), 0.1 ml of aliquot was poured on MRS (De Man, Rogosa and Sharpe) agar plates and incubated anaerobically at 37 °C for 24 hrs. Dense white colonies were formed on the surface of agar plates which were counted using a colony counter.
2.2. Identification of bacteria
Individual distinct colonies were sub-cultured and identified by colony morphology, Gram’s staining and biochemical tests (indole production test, methyl red test, Voges–Proskauer test, citrate utilization test, oxidase test, catalase test, starch hydrolysis test etc.) (Holt et al. 1994). Genomic DNA was extracted from isolated bacterial cultures (Green and Sambrook 2012), and PCR amplification of 16S rDNA gene was achieved by 704F (5'-GTAGCGGTGAAATGCGTAGA-3') and 907R (5'-CCGTCAATTCMTTTRAGTTT -3') (Madison et al. 2017). Sequencing of 16S rDNA was carried out at Xcelris Labs Limited, Gujrat, India using ABI 3730xl 96 capillary system using Big Dye Terminator v3.1 kit. The consensus sequence of the 16S rDNA gene was generated from forward and reverse sequence data using aligner software and EMBOSS merger. A BLAST search was performed with the 16s ribosomal RNA sequence database to find the closest homologous sequence. Based on the maximum identity score, first ten sequences were selected, and aligned using Clustal-W. The aligned nucleotide sequence was used to construct a phylogenetic tree using PhyML (Guindon et al. 2010; Nath et al. 2018). Geneious R8 software package was used to perform the above analysis (Biomatters Ltd., Auckland, New Zealand).
2.3. Assessment of probiotic properties
2.3.1. Test for resistance to low pH
Tolerance to pH is one of the essential attributes in in-vitro assays to determine the resistance to the acidic condition of the stomach. As the food remains in the stomach for at least 3 hrs (Thakkar et al. 2015); this time limit was taken under consideration for in vitro assay. pH tolerance was determined by inoculating 0.5 ml of 20 hrs old bacterial suspension to 5 ml of sterile phosphate buffer saline (PBS), adjusting the pH to 3 and 7.2, with 1N HCl. The sample was then incubated aerobically at 37 °C for 3 hrs, and their total viable count was measured at every 1 hr interval by spreading 100 µl of bacterial suspension on MRS agar plate. Optical density (OD 600 nm) was recorded at a regular interval to determine their viability and growth pattern (Hassanzadazar et al. 2012).
2.3.2. Simulated Gastric juice tolerance test
Overnight grown bacterial broth culture was taken and centrifuged at 5000 rpm for 15 min at 5 °C. The bacterial pellet was re-suspended in 10 ml PBS buffer and kept for 10 mins. Resistance to gastric juice and survival percentage was determined by incubating the isolates in simulated gastric juice and determining the viable cell counts at 1, 2 and 3 hrs.
Simulated gastric juice was prepared by using 3 g/l pepsin, 7 mM KCl, 45 mM NaHCO3 and 125 mM NaCl, adjusting at pH 3 (assay) and pH 7 (control) with 1M HCl and 1M NaOH respectively (Archer and Halami 2015).
2.3.3. Bile tolerance test
The bile tolerance test was conducted according to the method described by Gilliland et al. (1984). 100 µl of overnight grown bacterial culture was inoculated in MRS broth containing 0.3 % bile salts (Himedia Pvt. Ltd) and incubated at 37 ºC for 4 hrs. After the incubation period, 100 µl of the bacterial sample was spread onto MRS agar plate to determine the viability of bacteria in 0.3 % bile. Samples were also inoculated in MRS broth without bile, which acts as a control. Growth at a different time interval and percentage resistance of bacteria was determined by measuring the absorbance of MRS broth at 600 nm.
2.3.4. Pancreatin tolerance test
100 µl of 24 hrs old bacterial culture was inoculated in 10 ml of MRS broth containing 0.5 % (v/w) pancreatin and without pancreatin (control). Inoculated test tubes were kept in a shaker incubator for 48 hrs at 37 °C. Pancreatin tolerance was determined by comparing the viable cell count of test and control cultures in MRS agar plates (Khagwal et al. 2014). Pancreatin tolerance was also determined by measuring the OD (at 600 nm) at an interval of 0, 24 and 48 hrs.
2.3.5. Cell surface hydrophobicity
Adhesion is one of the important characteristics of probiotic bacteria. The ability of the bacteria to stick with hydrocarbons determines the extent of adhesion to the epithelial cells in the gastrointestinal tract known as cell surface hydrophobicity (Pan et al. 2006). 20 hrs old bacterial cultures were centrifuged at 12000 rpm for 5 mins at 4 °C. Pellets were washed twice by PBS buffer (pH 7.2) and re-suspended in 6 ml of PBS buffer. The initial absorbance was noted at 600 nm. After that, 3 ml of the bacterial suspension was mixed with 1 ml of hydrocarbons (n- Hexadecane and Toluene) and vortexed for 2 mins. The mixture was incubated and left undisturbed for 1 hr for phase separation. The aqueous phase was then removed carefully with a micropipette, and the final absorbance was measured at 600 nm (Rosenberg et al. 1980). The percentage of cell surface hydrophobicity was measured by the formula:
2.3.6. Cellular autoaggregation
Many bacteria have the ability to self-recognise surface structures and bind to themselves; this self-binding is known as autoaggregation or autoagglutination. The specific cell-cell interaction or autoaggregation was determined by the method described by Xu et al. (2009). Bacterial cultures were grown overnight at 37 °C and cells were harvested by centrifugation at 5000 rpm for 10 mins. Pellets were washed with PBS (pH 7.2), re-suspended in PBS buffer and the initial absorbance was noted at 600 nm. The suspension was incubated for 2 hrs at 37 °C. The final absorbance of the supernatant was measured at 600 nm. The percentage of cellular autoaggregation was measured by the formula:
2.3.7. Glucose fermentation test
Glucose fermentation test was conducted according to the method described by Gupta et al. (2011). 18 hrs old bacterial cultures were centrifuged at 4000 rpm for 15 min. Pellets were washed by PBS buffer and re-suspended in the PBS buffer. 500 µl of bacterial suspension was inoculated into MRS broth supplemented by 1 % glucose and 0.5 % phenol red as a dye and incubated for 24 hrs at 37 °C. Change in colour from purple to yellow indicates positive glucose fermentation, whereas no change in colour indicates negative glucose fermentation.
2.3.8. NaCl tolerance test
The growth of any bacteria can be affected by the amount of water entering or leaving the cell. If the medium has a low amount of solute, it is hypotonic and has high osmotic pressure on the cell. On the other hand, if the solute is high, the medium is hypertonic, and growth is considerably inhibited. In such cases, cytoplasm dehydrates and shrinks away from the cell wall. MRS agar plates were prepared in five different concentrations of NaCl. The concentration of NaCl was maintained at 0.5 %, 1 %, 5 %, 10 % and 15 % and one kept as a control (0 % NaCl). The MRS media was poured in Petri plates, bacterial strains were streaked on each plate and incubated at 37 °C for 24 hrs. The influence of NaCl concentrations on the degree of inhibition of bacterial growth was recorded.
2.4. Safety Assessment
2.4.1. Haemolytic Activity
Bacterial culture was streaked on sheep blood agar and incubated for 48 hrs at 37 ºC. The blood agar plate was observed and examined for signs of β-haemolysis (clear zone around the colonies), α-haemolysis (green-hued zones around colonies) or γ-haemolysis (no zones around the colonies) (Gerhardt et al. 1981).
2.4.2. Antibiotic susceptibility test
Antibiotic susceptibility test was performed by the Kirby-Bauer disk diffusion test (Baccer et al. 1966). Antibiotic discs were placed on freshly prepared lawns of each isolate on Mueller-Hinton agar (MHA) plates and incubated for 24–48 hrs at 37 °C. The diameter of the inhibition zones was measured, and the strains were classified following the standard antibiotic disc chart. Standard antibiotic discs were procured from ‘HiMedia’ which includes Gentamicin (120 µg), Vancomycin (30 µg), Tetracycline (30 µg), Polymixin-B (300 µg), Kanamycin (30 µg), Ofloxacin (5 µg), Co-Trimoxazole (25 µg), Meropenem (10 µg), Ceftriaxone (30 µg), Clindamycin (2 µg), Ampicillin (10 µg), Norfloxacin (10 µg), Rifampicin (5 µg), Amikacin (30 µg), Penicillin-G 10 µg), Cefdinir (5 µg), Ciprofloxacin (5 µg), Azithromycin (15 µg), Methicilin (5 µg), Streptomycin (10 µg).
2.4.3. Extraction of antibacterial agents and evaluation of their antagonistic activity
Isolation of antibacterial agents was performed by following the protocol of Hussein et al. (2018). 5 ml of 48 hrs old broth culture of test isolate GCC_19R1 was mixed with an equal volume of ethyl acetate and shaken well in a rotary shaker at 20 rpm for 10 mins. The isolates were centrifuged, and the supernatant was transferred into a centrifuge tube. Ethyl acetate was allowed to evaporate, and the remaining content was used to study the antagonistic effect on other bacterial isolates.
The effectiveness of bacterial metabolites was studied by well diffusion method (Shakhatreh et al. 2017). MHA plates were prepared, and wells were made using a sterile syringe puncture. The bacterial sample was spread on to the Petri plates using a sterile spreader, which includes Bacillus cereus strain SN_SA, Acinetobacter johnsonii strain SB_SK, Pseudomonas aeruginosa strain GCC_19W1, Stenotrophomonas maltophilia strain GCC_19W2, Cedecea davisae strain GCC_19S1 and Achromobacter spanius strain GCCSB1. Thereafter, wells were filled with test bacterial extracts, and plates were incubated for 24-48 hrs at 37 °C. The antibacterial activity was evaluated by measuring the zone of inhibition in mm.
2.5. Statistical analysis
All the above experiments were performed in triplicates, and the results were expressed as the means ± standard deviations of three independent replicates. The gathered data were analyzed using Microsoft Excel 2007 and SPSS version 16.