Isolation of bacteria from different honey sources
Honey samples were collected at the mature stage from different sources; Nigerian mountain honey (isolate 9A) and Acacia (Australia) (isolates 10A), New El-Wadi (Dakhla) Egypt (2M, 13M). Serial dilutions were made for each honey sample (10-9). Then spread on nutrient agar (NG) or MRS plates and incubated at 37 °C for 24h. One of the bacterial colonies was picked and purified by serial subculture and plating, then stored at −80 °C in the NG or MRSmedium containing glycerol (50%). Isolates were chosen for further morphological analysis and levansucrase activity.
Levansucrase assay was performed according to the method of Yanase et al. (1991). One unit of enzyme activity was defined as the amount of enzyme that produced sugars equivalent to 1 μmol of glucose/min.
Crude Levan (CL) precipitation
The levan yielding organisms were cultivated on the levan producing medium by shaking flask cultivation technique according to (Suhaimi et al. 2016). After the early stage of the stationary phase, the culture filtrate (CF) was centrifuged at 5000× g to get rid of bacterial cells. The CF was dialyzed against deionized water for 24 h by using a dialysis membrane (MWCO 14,000 Da, diameter 60 mm) to remove the unfermented sucrose, and any fermentation products had low molecular weights (MW). Levan was precipitated with three volumes of ice - cold ethanol (99%).
The isolated strains were examined by microscopic observation, and identification was based on morphological tests and 16s rRNA.
Identification of bacterial isolates by 16S rRNA gene sequencing
The 16S rRNA gene fragments of the four levansucrase producing bacteria were amplified using the universal primers F-27 (5′-AGAGTTTGATCMTGGCTCAG-3′) and R1494 (5′-CTACGGYTACCTTGTTACGAC-3′) using PCR machine (Bio-rad T100 thermal cycler). The PCR products were checked via agarose gel electrophoresis then purified using gel extraction kit and sequenced by Macrogenkoria.
Phylogenic analysis of bacterial isolates
The evolutionary history was inferred using the Neighbor-Joining method. The tree was computed using the Maximum Composite Likelihood method. The analysis involved 8 nucleotide sequences of which four sequences of 16S rRNA gene amplified from bacterial isolates of current study while the other four sequences are representing the most similar hits were obtained from the NCBI gene bank data base. Evolutionary analyses were conducted in MEGA5 software. The amplified region of the 16S rRNA segment from the isolates were submitted to NCBI Gen bank under Accession number: MT427639 of Bacillus subtilis (9A), Accession number: MT427640, of Achromobacter sp. (10A), Accession number: MZ349055 of Bacillus paranthracis (13M), Accession number: MW325728 of Bacillus paralicheniformis (2M).
Isolate’s cultivation was done on blood agar medium (Oxoid) supplied with 5% human blood and was grown at 37 °C for 24 h. Then incubated on Petri dishes containing their suitable medium nutrient agar (NG) for 9A and 10A or MRS for 2M and 13M at 37 °C for 24 h. Presence (or absence) of hemolysis was investigated visually (Chaiyawan et al. 2014).
The pattern of resistance/ sensitivity to the antibiotic of the isolated strains were tested using the disc diffusion method, as described previously. Four types of different antibiotic discs were used included Amoxicillin + Flucloxacillin, Ampicilin, Gentamicin and Benzathine benzaylpencillin. The tested isolates were activated for 24h on NG or MRS according to the isolate type. A total of 100 μL of the diluted cultures (adjusting the optical density for each strain to 0.1 O.D) was diffused in nutrient agar or MRS. The different antibiotic discs were applied on the surface. The plates were incubated at 4°C for 2h. and were incubated at 37 °C and 24 h of inoculation. The zone diameter of inhibition (ZDI) values was measured.
Resistance to low and high pH
The method of Conway et al. (1987) was used for the acid tolerance study of bacterial isolates. Where, the freshly prepared cultures were transferred into nutrient broth (NB) medium (5%) adjusted to pH 2.0 and pH 3.0 with 2 M HCl and to pH 9 and pH11 with 1 M NaOH. They were then incubated at 37 °C and culture samples were taken after 1, 3 and 6 h. Medium neutralization was done by serial dilutions in phosphate buffer (0.1 M, pH 7.0) and re-culture on nutrient agar (NG) OR MRS plates. The plates were incubated at 37 °C for 24 h and survival (%) was determined by comparing the viable bacterial count after incubation at pH 2.0, 3.0, 9 and 11 compared to the control bacterial count incubated at pH 7.
Bile salt resistance
Bile tolerance was conducted according to (Liong and Shah 2005; Westermann 2016) where the four isolates were grown overnight at 37 °C in MRS (2M,13M) or NG broth (9A, 10A) supplemented with 0.3 % (w/v) bile salt (Oxgall, USA). Then samples were incubated at 37 °C for 3 hr, 6 hr, 24 hr and slants without the bile salt were left as a control. Spectrophotometer (O.D. at 660 nm) was used to detect the bacterial growth. The ratios of bile salt resistance were calculated as follows: Percentage of surviving cells incubated with bile to the cell-count of control.
Antimicrobial activity of the isolates was carried out by agar well diffusion method (Mishra and Prasad 2005) using cell-free culture supernatants (CFCS) of the isolated probiotic strains against pathogenic indicator bacteria: Staphylococcus aureus, Bacillus cereus, Candida albicans NRRL Y-477, Asperillus niger NRRL 599 and Escherichia coli MC1400. Wells of 5 mm diameter were prepared and loaded with a volume with 100 μl of CFCS of each honey isolate and marked adequately with the isolates’ names. The plates were kept for two hours at room temperature, then incubated for 24 hours at 37 °C. The zone diameter of inhibition (ZDI) values was measured. The tests were performed in triplicate and the data were represented with mean ± SD.
Antioxidant activities and H2O2 tolerance
1.1‐diphenyl‐2‐picryl‐hydrazine (DPPH) assay
The free radical scavenging activity using the DPPH reagent was determined as described previously . The cells free supernatant of each isolate (100 µg) was added to 1.0 ml of freshly prepared methanolic DPPH solution (20 µg/ml) and stirred. The decolorizing processes were recorded after 5 min of reaction at 517 nm and compared with a blank control.
Antioxidant activity = [(control absorbance − sample absorbance) /control absorbance] × 100%
Tolerance of strains to H2O2 was assessed by the method of Li et al. (2012) but with only 30 min incubation time. Overnight grown cultures of the isolates were inoculated (1% v/v) into NG medium or MRS (control) and the tow medium containing 0.1% hydrogen peroxide and incubated at 37°C for 30 min. Probiotic criter
Cholesterol removal by tested bacterial isolates was determined according to the manufacture’s kits (Bio-diagnostic company for the diagnostic and research reagents).
Cholesterol oxidase assay
The activity of the extracellular CO enzyme was determined according to the method described by Inouye et al. (1982). Briefly, 0.1 mL of culture supernatant was added to 0.4 mL of 125 mM Tris-HCl buffer (pH 7.5). The mixture was incubated in a water bath at 37 °C. After 3 minutes, 25 µL of 12 mM of cholesterol in isopropanol solution was added to the mixture, and incubation proceeded for a further 30 minutes. Afterwards, 2.5 mL of absolute ethanol was added to the reaction medium, and then the amount of formed 4-cholesten-3-one was determined spectrophotometrically by measuring the absorbance at 240 nm. Reaction blanks were prepared by cholesterol solution with isopropanol. One unit of cholesterol oxidase activity (U) was defined as the amount resulting in the formation of 1 µmol of 4-cholesten-3-one in 30 minutes at 37°C. (concentration of 4-colesten-3-one was calculated from a standard curve previously prepared with serial dilutions (10–100 µg) of 4-cholesten-3-one dissolved in isopropanol.
One ml of the culture filtrate of each bacterial isolate was incubated separately at 37 °C with a mixture containing 1 ml of 0.1 M Tris-HCl buffer (pH 8.0), 2.5 ml deionized water and 3 ml of a vegetable oil. After 30 min, each test solution was transferred to a 50 ml Erlenmeyer flask and 3 ml of 95% ethanol was added to terminate the reaction. The formed fatty acids were titrated using 0.1 M NaOH and phenolphthalein as an indicator turning pink at the end point. Both test and blank were performed. Enzyme activity was expressed as units per ml enzyme (Sayali et al. 2013).