Sample collection, isolation and purification of Lactobacillus:
Multiple sets of milk products samples (Butter and cheese) were collected from local shops of Chittagong area in Bangladesh. For isolation of Lactobacillus, serial dilution (10-1 to 10-6) agar plate technique was used. Lactobacillus was purified by streak plate method on De Man Rogosa Sharpe (MRS) agar.
Test microorganisms:
The target pathogenic organisms used in this study were Staphylococcus aureus ATCC25923, Bacillus subtilis IFSTIM22, Pseudomonas aeruginosa CRL (ICDDR, B), E. coli ATCC25922, Salmonella typhii AE14296. The test microorganisms were standardized by using 0.5 McFarland standard. A 0.5 McFarland gives an approximate cell density of 1.5 × 108 CFU/mL, having an absorbance of 0.132 at wavelength of 600nm (Andrews et al., 2001). We used this standardization technique for all the necessary steps of this study.
Screening of isolated Lactobacillus for antibacterial activity:
MRS broth was used for antimicrobial metabolite production from Lactobacillus. 200 mL of MRS broth was autoclaved at 121 °C for 15 minutes and inoculated with the colonies of a Lactobacillus isolate and incubated at 37 °C for 2-3 days under stationary conditions. Then it was centrifuged (Model 6930, Kubota, Japan) at 9000 rpm for 15 minutes at 4 °C. The supernatant was then filtered through Whatman No. 1 filter paper to remove residual cells. Petri-plates were prepared by pouring sterile molten Muellar Hinton medium and allowed it to solidify. A hundred microliters of each standardized test microorganisms were spread on agar plates. Two wells (each 7 mm in diameter) made into agar plates with sterile borer. The wells were loaded with 100 μL of filtered LAB culture supernatant and 100 μL sterile broth. Plates were incubated at 37 °C for 24 hours. After incubation, the diameter of zone of inhibition was observed and measured (Pundir et al., 2013).
Identification of Lactobacillus isolates:
Lactobacillus species were identified based on their morphological characteristics including size and shape of the organism, arrangement of the cells, presence or absence of the spores, regular or irregular forms, acid fastness, gram reaction etc.; cultural and physiological characteristics including H2S production, nitrate reduction, deep glucose agar test, fermentation of different carbohydrates etc. All these characteristics were then compared with the standard description of "Bergey's Manual of Determinative Bacteriology", 8th edition (Buchanan and Gibbons, 1974).
pH tolerance and temperature sensitivity:
The Lactobacillus cultures were inoculated into sterile MRS broth tubes of varying pH, i.e., 2, 4, 7 and 9 and incubated at 37 °C for 24-48 hours. Another set of inoculated MRS broth was grown at varying temperatures, i.e., 27, 37, and 45 °C for 24-48 hours. The absorbance of MRS broths were taken at 600 nm by a spectrophotometer (Model T60U, pg instruments, UK) to measure microbial load (Rahman et al., 2019).
Bile salt and NaCl tolerance:
The MRS broth media with varying concentrations of bile salt (0.5, 1.0 and 2.0%) and NaCl (1, 3 and 7%) were inoculated separately with each Lactobacillus culture and incubated at 37 °C for 48 hours. Then the absorbance of MRS broths were taken at 600 nm by a spectrophotometer for measuring microbial load (Rahman et al., 2018).
Lactose utilization:
The acid production by Lactobacillus cultures was detected by observing the change in color of the medium. Sterilized fermentation medium (Peptone 10 g, NaCl 15 g, phenol red 0.018 g, lactose 5 g, for 1 L distilled water and final pH 7.0) was inoculated with Lactobacillus cultures and incubated at 37 °C for 24-48 h. Change in color from red to yellow indicates the production of acid (Ahmed and Kanwal, 2004).
Antibiotic susceptibility test:
The antibiotic susceptibility of Lactobacillus was assessed by using the Kirby-Bauer discs diffusion method on MRS agar plates. The used antibiotics were penicillin g (10 IU), chloramphenicol (30 μg), erythromycin (15 μg), cefixime (5 μg), cephradine (30 μg), streptomycin (10 μg) and rifamycin (5 μg) (Rahman et al., 2019).
Determination of bacteriocin production capability of the Lactobacillus xylosus: This experiment has been carried out according to the method described by Yang et al., 2012 (Yang et al., 2012). One milliliter of frozen Lactobacillus isolate was cultured 24 hours in 20 mL MRS broth. Then 1 mL culture was sub-cultured 24 h in 20 mL MRS broth. Cells were removed by centrifuging at 9000 rpm for 15 minutes. The supernatant was filtered through a sterile Whatman No. 1 filter paper and 100 μL of the pH unadjusted aliquot of cell-free supernatant (CFS) was added to the first well. The remaining CFS was adjusted to pH 6.0 with 1M/lN NaOH in order to rule out possible inhibitory effects due to organic acids. 100 μL of the pH adjusted CFS was filtered and added to the second well. The neutralized CFS was then treated with 1mg/mL of catalase (Merck KGa A, Germany) at 25 °C for 30 min to eliminate the possible inhibitory action of H2O2 and filtered. Then 10 μL catalase treated CFS was placed in the third well. If an inhibition zone were found in the third well, the isolates were able to produce bacteriocin or BLS. To confirm the production of a proteinaceous compound, CFS displaying antimicrobial production after acid neutralization and H2O2 elimination were treated with 1 mg/mL of proteolytic enzymes including papain and trypsin (Sigma-Aldrich Corporation, USA). 5ml of bacteriocin was taken in test tubes and treated with papain/trypsin (1 mg/mL) at pH 7. The test tubes with and without the enzyme (control) were incubated at 37 °C for 2 h and then heated at 100 °C for 3 min to denature the enzyme. Both the control and samples were assayed for antimicrobial activity by using agar well diffusion method (Rahman et al., 2019).