Sample preparation
Fresh water small fish, Ngasang (Esomus danricus), were smoked and sun dried until they crumbled. The petioles of an aroid plant, Khonagu (Alocasia macrorhiza) were cut into small pieces, washed with water and sun dried for an hour. The crumbled fish powder was crushed with plant material in a 1:1 ratio using a stone mortar and pestle to make a paste. The mixture was kneaded with clean hands to produce ball-shaped pieces, and fermentation was allowed by keeping the mixture at room temperature for 5–6 days in an earthen pot containing a thin layer of banana leaves. The ball-shaped pieces were taken out from the pot and mixed with onions and mustard oil. The mixture was kneaded again using a stone mortar and pestle and made into a ball shape. The ball-shaped pieces were kept again inside the earthen pot containing banana leaves for 2–3 days. The fermented non-salted fish product, Hentak, was brought to the laboratory for the bacterial isolation process. The involvement of fish in the experiments was approved by the Government of India Ethical Committee (IAEC-LC 05/13).
Isolation of lactic acid bacteria
One gram of Hentak was ground with sterilized distilled water using a mortar and pestle cleaned with ethanol (95 % w/v). The mixture was centrifuged at 8000×g for 15 min in order to remove heavy particles, and the supernatant was collected. The supernatant was serially diluted (10−1–10−5) for bacterial enumeration, and 1 ml of the suspension was poured onto sterilized MRS agar (g/l—proteose peptone 10.0, beef extract 10.0, yeast extract 5.0, dextrose 20.0, polysorbate 80 1.0, ammonium citrate 2.0, sodium acetate 5.0, magnesium sulfate 0.1, manganese sulfate 0.05, dipotassium phosphate 2.0, pH 6.5, Agar 18) plates. After spreading the suspension, the plates were incubated at 30 °C for 48 h. The total number of viable colonies was counted and expressed as colony forming units (CFU/ml). Based upon morphology, various colonies were selected for the isolation of pure bacterial cultures on MRS agar slants.
Bacteria of interest
Indicator bacterial strains (human enteric pathogens) were collected from the Department of Plant Biology and Biotechnology, Loyola College, Chennai, India. Both Gram positive (Staphylococcus epidermis MTTC 3615, Staphylococcus aureus MTCC 96, Enterococcus faecalis MTCC 439 and Micrococcus luteus MTCC 106) and gram negative (Shigella flexneri MTCC 1457, Yersinia enterocolitica MTCC 840, Enterobacter aerogens MTCC 111 and Proteus vulgaris MTCC 1771) bacteria were used for the present study. The indicator bacterial cultures were sub-cultured selectively onto basal media (nutrient broth for gram positive and Mueller–Hinton broth for gram negative bacteria, pH 7.0) at 37 °C for further study. A 24 h old bacterial culture was used for further experiments.
Assay for antibacterial substance production
The isolated lactic acid bacteria were screened individually for the production of antagonistic substances. The lactic acid bacteria were inoculated individually into sterilized MRS broth and incubated for 48 h at 30 °C. The indicator microorganisms were inoculated into Nutrient broth and Mueller–Hinton broth for 24 h at 37 °C and swabbed onto Mueller–Hinton agar (MHA) plates. Agar plates were punched using a sterilized, flamed and alcohol-dipped cork borer, and 5 mm wells were created. The lactic acid bacteria were centrifuged at 8000×g for 10 min, and the culture supernatant was subjected to membrane filtration (0.22 µm). The sterilized cell free supernatant was neutralized (pH 7.0) using 1 N NaOH in order to exclude the antibacterial effect of organic acids in the medium. The cell free neutralized supernatant (CFNS) was treated individually with catalase (Sigma, India; 1 mg/ml) and incubated at 37 °C for 2 h in order to eliminate the inhibitory effect of hydrogen peroxide. After catalase treatment, the CFNS obtained from lactic acid bacteria was then assayed for antibacterial assay against indicator bacteria using the agar well diffusion method. The growth inhibitory activity was expressed in arbitrary units (AU/ml). One AU was defined as the reciprocal of the highest level of dilution resulting in a clear zone of growth inhibition (Bhaskar et al. 2007).
Identification and molecular characterization of the isolate
The potent bacterium was identified using morphological and biochemical tests and further characterized using molecular tools. The genomic DNA of the potential isolate was isolated and purified using a QIAquick® kit (Qiagen Ltd., Crawley, UK). The amplicon sequencing was performed using universal primers 27F (5′ AGA GTT TGA TCG TGG CTC AG 3′) and 1492R (3′ GCT TAC CTT GTT ACG ACT T 5′). The 16S rRNA sequence of the isolate was subjected to BLAST, NCBI. Then, the sequence of the isolate was deposited into NCBI Genebank, and an accession number was assigned. The potential isolate was used for further experiments.
Media optimization
Lactobacillus pentosus strain LAP1 was inoculated individually into 250 ml conical flasks containing sterile production medium (50 ml) such as Nutrient broth, Mueller–Hinton broth, Luria–Bertani broth, MRS broth and Peptone broth to compare the production of antibacterial substances. The flasks were incubated at 30 °C for 48 h in an orbital shaker (120 rpm). The CFNS was obtained, and the arbitrary units (AU/ml) were estimated as described above against the indicator bacteria.
Optimization of culture conditions and medium components using the OFAT method
The suitable production media was optimized using various culture conditions (pH, temperature and inoculum volume) and medium components (carbon sources and nitrogen sources) utilizing the OFAT method after working out a series of experiments. The fermentation conditions and medium components were substituted one by one by keeping other factors constant in the production medium. The antibacterial substance production by strain LAP1 was examined by adjusting the pH (4, 5, 6, 7 and 8) of the production medium using 1 N HCl and 1 N NaOH. Similarly, the production of antagonistic substances with strain LAP1 was optimized by varying their respective conditions such as incubation temperature (20–70 °C) and inoculum volume (0.5–2 %) at the optimized pH. Likewise, the various media components such as carbon sources (maltose, fructose, sucrose, lactose, and xylose individually at 1.0 % w/v) and nitrogen sources (ammonium acetate, ammonium chloride, ammonium nitrate, ammonium sulphate and sodium nitrate individually at 0.5 % w/v) were substituted in the production medium in order to achieve maximum production of antibacterial substances. An appropriate control medium was also maintained. All of the flasks were aseptically inoculated with the isolate and kept in an orbital shaker (120 rpm) for 48 h. The CFNS was collected after centrifugation at 8000×g for 10 min, followed by membrane (0.22 μm) filtration of the supernatant and neutralization. The CFNS was collected and the antibacterial activity (AU/ml) was examined against the most susceptible indicator bacteria as described above.
Effect of supplements on antibacterial substance production
The production of antibacterial substances by L. pentosus strain LAP1 was assessed under optimized culture conditions in the suitable production medium supplemented with Tween20 (1 % v/v), Tween40 (1 % v/v) Tween80 (1 % v/v) and glycerol (1 % v/v). An appropriate control medium was also maintained, and the antagonistic activity of CFNS was determined as described above using the most susceptible indicator organisms.
Characterization of CFNS
The CFNS from strain LAP1 was characterized with respect to pH, heat treatment and proteolytic enzymes. The stability of CFNS at different pH values (pH 3, 5, 7, 8 and 10) was tested by adjusting the pH of the supernatant with either 1 N HCl or 1 N NaOH. The adjusted supernatants were incubated for 4 h at room temperature, and the activity was calculated using indicator bacteria. The CFNS of the isolate was subjected to heat treatment at temperatures of 60 °C for 60 min, 100 °C for 30 min, and autoclaving (121 °C/15 min). CFNS and H2O2-eliminated CFS (cell-free supernatant) without any heat treatment served as a control. Aliquots of each treatment were taken after the required incubation period, and the activity of heat treated CFNS was determined against indicator bacteria as described earlier using the agar well diffusion method. Similarly, the sensitivity of inhibitory substances produced by the isolate to proteolytic enzyme such as pepsin (1 mg/ml) was determined. The reaction mixtures were then incubated at 37 °C for 1 h, and the antagonistic activity of the supernatant was determined as described above.
Determination of DPPH free radical scavenging activity
The DPPH (2,2-diphenyl-1-picrylhydrazyl) assay is one of the most commonly used methods to detect free radical scavenging activity. The DPPH scavenging assay for the CFNS of strain LAP1 was measured by the method of Chen et al. (2005) with some modifications. Various concentrations (100–1000 µl) of CFNS were mixed with 1 ml of 0.05 mM DPPH solution. The reaction was incubated in the dark at room temperature for 30 min. DPPH solution was used as a control, and a combination of CFNS and methanol was used as the blank. The DPPH scavenging capacity of the CFNS of the isolate was calculated by measuring the decrease in absorbance at 517 nm compared to the control. The DPPH scavenging capacity was calculated as:
$${\text{DPPH scavenging capacity (\% )}} = \left[ {{{\left( {{\text{A}}_{{{\text{sample}}}} - {\text{A}}_{{{\text{blank}}}} } \right)} \mathord{\left/ {\vphantom {{\left( {{\text{A}}_{{{\text{sample}}}} - {\text{A}}_{{{\text{blank}}}} } \right)} {{\text{A}}_{{{\text{control}}}} }}} \right. \kern-\nulldelimiterspace} {{\text{A}}_{{{\text{control}}}} }}} \right] \times 100$$
Statistical analysis
All of the experiments were performed in triplicate, and the data were calculated as the Mean ± SD with MS-Excel.
