Isolation of lactic acid bacteria from fermented food products
Lactic acid bacteria were isolated from yoghurt, curd, dosa batter, idli batter and soaked and ground rice batter by weighing 1g of sample and serially diluted with 0.85% NaCl. After homogenization, 10-2 serial dilution of samples were spread on MRS agar medium substituted with bromocresol green (0.04 g/l) and incubated at room temperature (30±2ºC) for 24-72 h aerobically and an aerobically. For anaerobic incubation anaerobic jar was used. Randomly picked colonies with yellow colour zone were streaked repeatedly on MRS agar and incubated at room temperature (30±2ºC) for 24 h to obtain pure colonies. Isolated pure colonies were streaked on MRS agar substituted with 1% cycloheximide to check present of yeast among isolates (Yelnetty et al 2014). The isolated pure colonies were stored in MRS agar slant for further studies. Gram staining test was performed to all the isolated colonies. Gram positive colonies were selected to perform antibacterial activity assay.
Detection of antibacterial activity of lactic acid bacteria
Single colony from the selected bacterial culture by MRS agar containing bromocresol green substituted with 1% cycloheximide was incubated at room temperature (30±2ºC) for 24 h in MRS broth for fermentation. After fermentation, broth was centrifuged at 13000 rpm for 15 min. and supernatant was collected and filtered through 0.45 µm syringe filter. Resulted supernatant was assayed for detection of antibacterial activity against food spoilage organisms. The culture of those food spoilage organisms were obtained from Faculty of Science, University of Jaffna, Sri Lanka. Food spoilage organisms (Staphylococcus aureus, Enterococcus faecalis, E.coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Serratia marcescens, Salmonella sp., Proteus sp., Micrococcus sp., and Bacillus sp.,) were streaked on the nutrient agar and incubated at 37ºC for 24 hours. Inocula size of food spoilage organism was adjusted by using heamocytometer at 6.0×108cfu/ml. 100 µl of the 24 h old culture suspension of 6.0×108 cfu/ml indicator strains were mixed with 5 ml of nutrient agar earlier cooled to 47°C and then poured onto already solidified nutrient agar (10 ml) plates and allowed to solidify. After solidification, 10 mm wells were cut by using sterile cork borer. The 100 µl of crude supernatants were added to the wells and incubated at 37ºC for 24 hours. Diameter of clear zone around the well was measured by using digital Vernier caliper. The potential bacterium was selected based on wide spectrum inhibition activity.
Physiological characterization of selected bacterial strains
Temperature tolerance test
Abilities of the selected strains at different temperature range from 15ºC to 55ºC were evaluated. Selected strains were inoculated into nutrient broth and kept at different temperature. Turbidity was observed after 5 days.
pH tolerance test
Nutrient broth was prepared at pH values 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and 11. Twenty four hours old bacteria cultures were inoculated into respective nutrient broth in test tubes and incubated at37oC for 5 days. Nutrient broth without inoculum was used as control. Turbidity of the nutrient broth after 5 days was observed with control.
NaCl tolerance test
Nutrient broth substituted with different concentration of NaCl ranging from 1%-16% was prepared. 24 hours old bacteria cultures were inoculated into respective nutrient broth in test tubes and incubated at 37oC for 5 days. Nutrient broth with different NaCl concentration without inoculum was used as control. Results were obtained by observing turbidity of nutrient broth after 5 days and compared with control.
Bile tolerance test
Abilities of the selected strains to tolerance for different bile concentration such as 0.3%, 0.5% and 0.8% were investigated by inoculate 200 µl of 24 h old 1.5×108 cfu/ml of inoculum of each isolate into the nutrient broth substituted with different concentration of bile. Inoculated medium was incubated at 37ºC for 5 days and turbidity was observed.
Biochemical characterization of selected bacterial strains
Selected strain was subjected to gram staining, catalase test, motility test (hanging drop technique) and endospore staining initially.
Gas production from glucose and fructose
Ability of strains to produce gas was evaluated by inverting Durham tube into test medium. Test medium was prepared by incorporating sugar (5g/l), peptone (10g/l) and NaCl (5g/l) and phenol red (0.018g/l) at pH 7.0.
Fermentation of different carbohydrate
Acid production of strains from various carbon sources namely arabinose,fructose, galactose, glucose, lactose, maltose, mannitol, mannose, raffinose, ribose,sorbitol, starch, sucrose and xylose were investigated. The medium with different sugar was prepared by incorporating sugar (5g/l), peptone (10g/l) and NaCl (5g/l) and phenol red (0.018g/l) at pH 7.0.
Arginine Dihydrolase test (ADH)
ADH test medium was prepared by incorporating peptone (1g/l), NaCl (5g/l), K2H2PO4 (0.3g/l), Arginine (10g/l) and bromocresol purple (0.016g/l) and pH was adjusted to 6.2. Selected strains were inoculated into each tubes incubated at 37ºC for 5 days. Uninoculated test tube with medium was considered as negative control. Colour changes of medium were observed at every day up to 5 days.
Gelatin hydrolysis test
Gelatin hydrolysis test medium was prepared by incorporating peptone (5g/l), beef extract (3g/l) and gelatin (120g/l) and pH was adjusted to 6.8.
Citrate utilization test
Citrate utilization medium was prepared with composition of sodium citrate (2g/l), MgSO4 (0.2g/l), ammonium dihydrogen phosphate (1g/l), dipotassium phosphate (1g/l), sodium chloride (5g/l), bromothymol blue (0.080g/l) and Agar (15g/l) and pH was adjusted to 6.8.
Molecular characterization of isolated bacterial strains
Bacterial DNA was extracted by Wizard® Genomic DNA Purification Kit and quantified by Quantus Flurometer. DNA region of 16S, 23S rRNA ISR and its flanking 23S rRNA was PCR amplified and sequenced.