In Vitro Probiotic Potential and Safety Evaluation (Hemolytic, Cytotoxic Activity) of Bifidobacterium Strains Isolated from Raw Camel Milk

The present study was designed to isolate Bifidobacterium strains from raw camel milk and to investigate their probiotic characteristics. Among 35 isolates, 8 were identified as Gram-positive, catalase negative, non-spore forming, non-motile and V or Y shaped rods. B-2, B-5, B-11, B-19 and B-28 exhibited good survival at low pH and high bile salt concentration. Most of the isolates were resistant to nalidixic acid, fusidic acid, polymyxin B, neomycin, streptomycin, gentamicin, rifampicin and kanamycin. Furthermore, the production of exopolysaccharides (EPS), adhesion characteristics, antioxidant properties, antagonistic activities, nitrite reduction and cholesterol assimilation were also studied. Isolate B-11 was chosen because it exhibited most of the probiotic properties among all the tested isolates. It is identified as the member of Bifidobacterium longum group through 16S rRNA gene sequencing and named as B. longum B-11. B. longum B-11 was further selected for in vivo attachment to rat intestine and scanning electron micrographs revealed that attachment of a large number of rods shaped bacterial cell. Our findings suggest that B. longum B-11 processes excellent attributes to be used as potential probiotic in the development of functional probiotic food.


Chemicals
All media, chemicals, reagent and kits used in this study were of analytical grade and purchased from St. Louis, MO, USA., Sigma-Aldrich Chemicals Ltd., Merck and Co. Inc. and White House Station, NJ, USA.

Sample Collection and Isolation
Forty-five raw camel milk samples were collected from local lactating healthy camels (Camelus dromedaries) from different regions of Punjab, Pakistan. The samples were collected into a sterilized container. Due to long-distance, samples were placed in iceboxes until delivered to the Food Microbiology and Biotechnology Laboratory, NIFSAT, UAF and immediately analyzed upon arrival. Samples were homogenized (10% w/v) in sterile phosphate buffer using stomacher. After preparation of dilutions in sterile saline (0.85% w/v NaCl), the samples were plated on de Man Rogosa Sharpe (MRS) agar (Difco, Sparks, MD, USA) supplemented with L-cysteine 0.05% (w/v) (Sigma Aldrich, St. Louis, MO, USA). Plates were incubated anaerobically in an anaerobic chamber (Bactron Shel Lab II-1, Sheldon Manufacturing Inc., Cornelius, OR, USA) at 37 • C for 48 h. After incubation, morphologically dissimilar colonies were selected and re-streaked on an agar plate to get pure isolates.

Preliminary Identification and Screening of Bifidobacterium
All isolates (35) were analyzed on the basis of morphology, Gram staining, catalase test, motility test and endospore test. After preliminary screening, 8 isolates were identified as a member of genus Bifidobacterium on the basis of Gram-positive, catalase-negative, non-spore forming, non-motile and V or Y shaped rods. These isolates were further analyzed for growth characteristics i.e., growth at various temperatures (10 • C, 37 • C and 45 • C), growth at different NaCl concentrations (3%, 5% and 7%) and growth at various pH (4.4 to 9.6). These isolates were also analyzed for their ability to produce CO 2 and ferment various sugar by using API 50CH kit. Microscopic examination was performed by using the EVOS FL Auto Cell Imaging System (Thermo Fisher Scientific, Waltham, MA, USA). The pure isolate was preserved in MRS broth in 30% glycerol at -80 • C as a frozen stock. The culture was reactivated prior to use by sub-culturing at least 3 times in MRS broth.

Survival under Gastrointestinal Conditions and Phenol Tolerance
Isolates were screened for their ability to withstand in harsh gastrointestinal conditions i.e., low gastric pH, bile salt and 0.4% (w/v) phenol. Isolates were cultivated in MRS broth to get the desired cell number (10 9 logs CFU/mL) after 18 h of incubation at 37 • C. The simulated gastric juice (SGJ) containing pepsin (3 g/L) was prepared in sterile saline (0.85%, w/v NaCl) and adjusted the pH 2, 3 by using 1 M HCl. 1 mL (10 9 logs CFU/mL) of overnight grown cell suspension was mixed with 9 mL of sterilized SGJ under aseptic conditions and incubated at 37 • C for 2 h. Similarly, tolerance to bile salt and phenol was evaluated by inoculating bacterial suspension (1 mL containing 10 9 logs CFU/mL) in MRS broth tubes containing a various concentration of bile salt (0.1%, 0.5% and 1% w/v) and phenol (0.4%, w/v) [24]. After incubation, the viable count was determined by plating and expressed as CFU/mL [25].

Production of Exopolysaccharides (EPS)
The EPS production was determined as described by Liu, et al. [26]. 1% (v/v) of each isolated bacterial culture (10 9 CFU/mL) was inoculated into MRS broth supplemented with 2% (w/v) glucose and incubated (37 • C for 24 h). After completion of incubation, cell pellets were removed by centrifugation (4000× g, at 4 • C for 10 min). Trichloroacetic acid 4% (w/v) was added and mixture was vortex at 4 • C for 3 h. Precipitated proteins were removed through centrifugation (22,000× g, 20 min), supernatant was concentrated through evaporation. Four volumes of ethanol (95%, v/v) was added to precipitated EPS, the mixture was centrifuged (22,000× g, at 4 • C for 20 min) and stored for 24 h at 4 • C. EPS was dialyzed (6000 Da to 8000 Da molecular weight) for 48 h at 4 • C and freeze-dried to make powder. The EPS amount was calculated by following the phenol sulfuric acid method [27].

Auto-Aggregation Assay
The auto-aggregation assay was accomplished by following the method as described by Xu, et al. [28] with little modifications. Overnight culture of all the tested isolates were collected after centrifugation (4000× g, at 4 • C for 10 min). The cells were washed two times with phosphate buffer saline (PBS) and re-suspended in PBS to reach an absorbance of (0.5 ± 0.02) at 600 nm at 0 h (A 0 ). Then, 1 mL of each bacterial suspension was vortexed for 5 sec and incubated (37 • C for 2 h, 6 h, 12 h and 24 h). Supernatant absorbance was measured at 600 nm after 2 h, 6 h, 12 h and 24 h of incubation (A 2 ) of incubation (Equation (1)).
Whereas, A 0 = Initial absorbance at 0 h; A 2 = Final absorbance after 2 h, 6 h, 12 h and 24 h of incubation

Cell Surface Hydrophobicity
Cell surface hydrophobicity was evaluated via all the 8 isolated bacteria that have the ability to bind with hydrocarbons by following the method as described by Kotzamanidis,et al. [29] with slight modifications. Briefly, overnight grown cells were collected after centrifugation (4000× g, at 4 • C for 10 min). The cells were washed two times with PBS having pH 7 and resuspended in PBS to reach an absorbance of (0.5 ± 0.02) at 600 nm at 0 h (A 0 ). After that, 1 mL of hydrocarbon (xylene) was mixed separately with 3 mL cells suspension and pre-incubated at 37 • C for 10 min. The cell suspension and hydrocarbon mixture were vortexed for 2 min and kept for 20 min for phase separation (water and hydrocarbon phase). After the collection of the aqueous phase, absorbance was measured at 600 nm (A 1 ) (Equation (2)).
Whereas, A 0 = Initial absorbance, A 1 = Final absorbance 2.4.5. DPPH Free Radical Scavenging Activity DPPH free radical activity of isolates was analyzed by following the method described by Chen, et al. [30]. Briefly, 100 µL of freshly prepared cells (10 9 CFU/mL) was mixed with 1 mL of (0.05 mM) DPPH solution. The mixture was mixed and kept for 30 min in a dark place. The absorbance was recorded at 517 nm and DPPH scavenging activity was calculated as given in (Equation (3)). The resistance of isolates to hydrogen peroxide was determined by a modified method described by Oberg, et al. [31]. 10 mL of freshly prepared culture were mixed with 10 mL 0.85% (w/v) NaCl in different test tubes with varying concentrations of H 2 O 2 (0.5, 1.0 and 1.5 mM). The viability of isolates was determined by withdrawing samples after 60 min of incubation.

Depletion of Sodium Nitrite
The depletion of sodium nitrite by isolates was documented by following the method as described by Wu, et al. [32] with minor changes. 1 mL (1500 µg/mL) of sterilized sodium nitrite solution was mixed with 9 mL of MRS broth (pH 6.5) to making it 150 µg/mL. 100µL freshly prepared bacterial culture (10 9 CFU/mL) was inoculated and incubated anaerobically for 48 h at 37 • C. In the case of control samples, sterile water was used instead of inoculum. The colorimetric nitrite method was used to measure initial and final absorbance at 538 nm by following the method described by Yan, et al. [33]. 5 mL of a mixture containing (inoculum, MRS broth, nitrite) was deproteinated and defatted by using 10 mL of ZnSO 4 (0.42 mol/L), then the mixture was filtered. 1 mL of each these three-color developing solutions (0.2% (w/v) sulfanilamide, 0.1% (w/v) N-1-naphtyethylene diamine dihydrochloride and 44.5% (v/v) HCl) were added in filtrate and mixed it well. The mixture was kept in dark place at 37 • C for 5 min. The optical density of the color mixture was recorded at 538 nm against a blank. 3000 µg/mL standard sodium nitrite solution was prepared and a standard curve was constructed by following the same protocol (Equation (4)).
Whereas, C i = Amount of nitrite present in MRS broth at 0 h; C f = Amount of nitrite present in MRS broth after 48 h.

Antibacterial Activity
Antibacterial activity of isolates against three human pathogens i.e., Escherichia coli ATCC 25922, Salmonella typhimurium ATCC 14,028 and Staphylococcus aureus ATCC 25,923 were determined through ager well diffusion method [10]. 1 mL of the freshly prepared cells of each isolate was filtered by using a 0.2 µm syringe filter. Each indicator pathogen was overlaid on Muller Hinton agar plates. 50 µL of culture filtrate (cell-free supernatant) was poured into 7 mm diameter wells created with borer. The plates were incubated at 37 • C for 24 h. The clear zone diameter of each isolate against each indicator pathogen was measured after incubation. The diameter of clear zone was described as: no inhibition (0 mm), diameter between 0-3 mm (weak inhibition), diameter between 3-6 mm (good inhibition) and diameter greater than 6 mm (strong inhibition) [10].

Cholesterol Reduction Assay
Cholesterol stock solution was prepared by dissolving 30 mg of water-soluble cholesterol (polyoxyethanyl-cholesterol sebacate) in 10 mL distilled water and filtered (0.45 µm). MRS broth containing 100 µL/mL cholesterol stock and 0.30 g/100 mL bile salt (oxgall) was inoculated with 1% of freshly prepared culture and incubated (37 • C for 24 h). The samples were withdrawn after a predetermined time interval (6,12, and 24 h) and centrifuged (4000× g, at 4 • C for 10 min). The supernatant was used to determine cholesterol content by using modified colorimetric method Rudel and Morris [34] with slight modification [35]. 2 mL of this supernatant was mixed with 2 mL (33% w/v) KOH and 2 mL ethanol (96%). This mixture was vortexed for 2 min, incubated at 37 • C for 30 min and cooled to room temperature. After co40oling, 3 mL of hexane and 2 mL of Milli-Q water was added and vortexed for 2 min. The mixture was kept for separation of two phases, hexane layer was separated (upper layer) and evaporated. 2 mL of o-phthalaldehyde reagent (50 mg OPA dissolved in 100 mL glacial acetic acid) was added and vortexed for 2 min. 0.5 mL H 2 SO 4 (98% purity) was added into this mixture and vortexed. After 10 min rest at room temperature, absorbance was recorded at 550 nm. Cholesterol removal (%) was calculated as given in Equation (5).

Antibiotic Susceptibility
The antibiotic susceptibility of isolates was tested against selected antibiotics (ampicillin, cephalothin, chloramphenicol, cloxacillin, erythromycin, nalidixic acid, fusidic acid, polymyxin B, neomycin, streptomycin, gentamicin, rifampicin, kanamycin, novobiocin, penicillin, spectinomycin, tetracycline and vancomycin) by using antibiotic disc diffusion method on MRS agar plates as described by Vijayakumar, et al. [36]. MRS agar plates were prepared by swabbing the overnight culture of tested isolates. Antibiotic discs were placed on solidified MRS agar and give them 30 min for antibiotic diffusion and after that incubated (37 • C for 48 h). The zone of inhibition was measured for each antibiotic disc after the completion of incubation. The antibiotic susceptibility was differentiated by measuring zone of inhibition i.e., <8 mm resistant, 8-10 mm moderate and >10 mm susceptibility.

Hemolytic Activity
The hemolytic activity of isolates was determined by using Columbia agar containing 5% (w/v) sheep blood and the plates were incubated at 37 • C for 48 h. After incubation, the hemolytic activity of isolated strains was evaluated and classified on the basis of lysis of red blood cells in the medium around the colonies. The green zones around colonies (α-hemolysis), clear zones around colonies (β-hemolysis) and no zones around colonies (γ-hemolysis) on Columbia blood agar plates. Only strains with γ-hemolysis are considered as safe [37].

Cytotoxicity
In vitro cytotoxicity activity isolates were determined according to the procedure reported in Mohanty, et al. [38] with slight modification. The cytotoxic (anti-cancer) activity of isolated strains was carried out using 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. This assay measured the development of blue formazan product as an MTT reduction by mitochondrial dehydrogenase, which is the indicator of cell viability and normal functioning of mitochondria. Briefly, exponentially growing 3 × 10 4 Caco-2 cells/well (100 µL/well) were incubated in 96-well plates and cultured for 24 h. The culture medium was removed after 24 h and each tested isolate (10 7 CFU/mL) cell-free filtrate was added to the culture medium. The Caco-2 cells were further incubated for 8 h and 24 h. After incubation, the percentage of cells surviving was counted using MTT assay. MTT solution (100 µL/mL) was added to each well. After 2 h of incubation at 37 • C, 100 µL of dimethylsulfoxide (DMSO) was added to dissolve the blue crystals and absorbance was read. The optical density was measured at 570 nm using a microplate reader.

Molecular Identification of Isolate (B-11)
The selected potential probiotic Bifidobacterium isolate (B-11), that possessed most of the probiotics functional characteristics were identified on the basis of 16S rRNA gene sequencing. Bacterial genomic DNA was extracted by following the phenol-chloroform extraction method as described by Martinez, et al. [39] and PCR was performed by using universal 16S primers (8F and 1391R). Amplicons were electrophoresed using 1% agarose gel and purified by QIAquick PCR purification kit (Qiagen, USA) and quantified using Nano Drop (ND-1000) Spectrophotometer. The gene sequencing was done by the Genomics Core Facility at Michigan State University, USA. The obtained sequence was compared and molecularly identified with the available nucleotide database from the NCBI GenBank using the BLAST search (https://blast.ncbi.nlm.nih.gov/Blast.cgi). After gene sequencing, the pure isolate was labeled as Bifidobacterium longum B-11. The obtained sequence was deposited into NCBI GenBank and allotted NCBI accession number (MH041649.1).

Attachment of B. Longum B-11 to Rats Intestine; In Vivo Studies
In vivo studies were carried out on 10 Sprague Dawley rats (age: 3-4 weeks; weighing 15-22 g). The rats were accommodated in the animal room of the National Institute of Food Science and Technology (NIFSAT), University of Agriculture, Faisalabad after obtaining ethical approval granted from the head of NIFSAT, University of Agriculture, Faisalabad Pakistan. During the entire period, the environment in the animal room was maintained (temperature 23 ± 2 • C; relative humidity 55 ± 5%) with a 12 h light/dark cycle. The animals were fed on normal diet and tap water ad libitum for one week before the start of the trail for adaptation and to confirm their normal growth behavior. Before the start of the experiment, animal feed and water intake were observed to be free from any contamination and pathogenic infection. Rats were randomly divided into two groups (G 0 and G 1 ), having 5 rats in each group. G 0 group were administrated with normal diet while G 1 were administrated B. longum B-11 (10 7 -10 9 CFU/mL) as a single dose along with normal diet for a period of one week. Administration was achieved through intragastric gavage.

Scanning Electron Microscopy of Rats Intestine
After one week of oral administration of B. longum B-11, the Sprague Dawley rats were sacrificed by cervical dislocation and their intestines were removed gently. The segmented intestine was opened and washed with PBS and fixed in 4% glutaraldehyde for 1 h [40]. The dehydrated samples were placed on aluminum stubs and coated with gold-palladium [41]. Field emission scanning electron microscope (FE-SEM) (S4700, Hitachi High-Technologies Corporation, Japan) was used to observe the attachment of B. longum B-11 on the intestinal surface and micrographs were taken with the same microscope.

Statistical Analysis
All the experimental trials were carried out in duplicate and for each trial, analysis was performed in triplicate. The results were presented as mean ± standard deviation (SD). The data collected in this study were analyzed statistically through a completely randomized design (CRD) using Statistix 8.1. Level of significance (p < 0.05) was estimated by using the analysis of variance technique (ANOVA) with two factor factorials under CRD followed by Tukey's multiple comparison test.

Ethical Approval
All procedures performed in studies involving animals were in accordance with the ethical standards of the institution or practice at which the studies were conducted.

Isolation and Identification of Presumptive Bifidobacterium Isolates
A total of 35 isolates (Bifidobacterium and LAB) from raw camel milk were preliminary identified. Out of these, eight isolates (B-2, B-5, B-11, B-14, B-19, B-21, B-28 and B-33) showed an appearance of bifidobacteria on the MRS medium and selected for a further experiment on the basis of cultural, morphological, microscopic, physiological and biochemical characteristics. Most of the isolates formed white to off white, shiny round colonies on an agar plate. Preliminary characterization suggested that selected isolates were all Gram-positive, rods (V or Y) shaped, catalase-negative, non-motile and non-spore forming (Table 1).

Survival under Gastrointestinal Conditions and Phenol Tolerance
Isolated strains have the ability to survive at pH 2 and pH 3 during 2 h of incubation (Figure 1a). At pH 2 the survival rate was lower as compared to pH 3 for all the tested isolates. The survival rate of B-2, B-5, B-11, B-19 and B-21 remained above 60% at pH 2 and pH 3 during 2 h of incubation.
Results also revealed that B-5, B-11 and B-19 showed significantly (p < 0.05) high resistance to gastrointestinal conditions (75%, 85% and 77%, respectively) at pH 2. All the isolates have the ability to tolerate various concentrations of bile salt (0.3%, 0.5%, and 1%) during 3 h of incubation ( Figure 1b). Maximum survival was observed at 0.3% bile salt concentration while the least was observed at 1% bile salt concentration. As the concentration of bile salt increases the survival rate decreases. The strain B-11 survived well even such a high concentration of bile salt (1%) than any other strain. The potential of isolates to tolerate 0.4% phenol is shown in Table 2. All the isolates have the ability to tolerate 0.4% phenol after 24 h of incubation. Result revealed that non-significant difference was observed in the viable cell count after 24 h of incubation with minimum log reduction (<1 log CFU/mL).

Production of Exopolysaccharides (EPS)
The potential of isolates to produce EPS was shown in Table 2. All the isolates were able to produce EPS but each strain exhibited different EPS production capacities. All the tested isolates had the ability to produce exopolysaccharides when grown on MRS agar supplemented with 2% (v/v) glucose as a carbon source. Among the isolates, B-11 produced the highest EPS (123 mg/L) followed by B-5 (112 mg/L) and B-21 (102 mg/L), while the least production was observed in B-19 (56 mg/L) and B-28 (61 mg/L).

Auto-Aggregation Assay
Cell surface traits (auto-aggregation and cell surface hydrophobicity) of probiotics are imperative for bacterial colonization and protection. All the tested isolates had high auto-aggregation activity. The isolate B-11, B-21 showed maximum auto-aggregation (65% and 60%) while the least was observed in B-33 (44%). However, auto-aggregation increases as the incubation time increases, so all the isolates showed an increase in auto-aggregation after 24 h of incubation ( Figure 2).
The potential of isolates to produce EPS was shown in Table 2. All the isolates were able to produce EPS but each strain exhibited different EPS production capacities. All the tested isolates had the ability to produce exopolysaccharides when grown on MRS agar supplemented with 2% (v/v) glucose as a carbon source. Among the isolates, B-11 produced the highest EPS (123 mg/L) followed by B-5 (112 mg/L) and B-21 (102 mg/L), while the least production was observed in B-19 (56 mg/L) and B-28 (61 mg/L).

Auto-Aggregation Assay
Cell surface traits (auto-aggregation and cell surface hydrophobicity) of probiotics are imperative for bacterial colonization and protection. All the tested isolates had high auto-aggregation activity. The isolate B-11, B-21 showed maximum auto-aggregation (65% and 60%) while the least was observed in B-33 (44%). However, auto-aggregation increases as the incubation time increases, so all the isolates showed an increase in auto-aggregation after 24 h of incubation ( Figure 2).  Table 2).

Resistance to Hydrogen Peroxide
All the tested isolates have the ability to resist H2O2. Results revealed that a significant impact on the viability of isolates was observed as the concentration of H2O2 increases exhibiting a decreasing trend. B-11 showed that the maximum viable cells count (8.1 log CFU/mL) while the least was observed in B-33 (5.45 log CFU/mL) at 1.5 mM H2O2 (Figure 3). In the case of B-2, B-5, B-14, B-19 and B-21 the viable cell count was >10 6 log CFU/mL.

Depletion of Sodium Nitrite
Nitrite is used as an additive in a number of fermented products. It is still considered undesirable in food products due to its safety concerns. It is recognized as a carcinogenic and teratogenic substance due to its high oxidative and reductive activities. There is a need to eliminate nitrite during fermentation. The results propounded in Table 2 revealed that all isolates had the ability to deplete nitrite. Among the isolates, B-11 depleted maximum nitrite 84% followed by B-5 (76%) while the least nitrite depletion was observed in B-21 (50%).

Antibacterial Activity
The results of the antibacterial activity as a halo of growth inhibition produced on agar plates by tested isolates against indicator pathogens were presented in Table 3. Isolates exhibited strong antimicrobial activity against pathogenic bacteria (Escherichia coli, Salmonella typhimurium and Staphylococcus aureus). Most of the isolates inhibited indicator pathogenic bacteria and the zone of inhibition was >6 mm. The isolates B-19 and B-28 exhibited zero inhibition against Escheria coli and Salmonella typhimurium, respectively. The observed inhibitory property of the isolates could be attributed due to the production of antimicrobial compounds. Based on the results, E. coli and Salmonella typhimurium were the most sensitive and S. aureus was the most resistant indicator bacteria against the tested isolates. Table 3. Antibacterial activity of isolates (means ± SD).

Depletion of Sodium Nitrite
Nitrite is used as an additive in a number of fermented products. It is still considered undesirable in food products due to its safety concerns. It is recognized as a carcinogenic and teratogenic substance due to its high oxidative and reductive activities. There is a need to eliminate nitrite during fermentation. The results propounded in Table 2 revealed that all isolates had the ability to deplete nitrite. Among the isolates, B-11 depleted maximum nitrite 84% followed by B-5 (76%) while the least nitrite depletion was observed in B-21 (50%).

Antibacterial Activity
The results of the antibacterial activity as a halo of growth inhibition produced on agar plates by tested isolates against indicator pathogens were presented in Table 3. Isolates exhibited strong antimicrobial activity against pathogenic bacteria (Escherichia coli, Salmonella typhimurium and Staphylococcus aureus). Most of the isolates inhibited indicator pathogenic bacteria and the zone of inhibition was >6 mm. The isolates B-19 and B-28 exhibited zero inhibition against Escheria coli and Salmonella typhimurium, respectively. The observed inhibitory property of the isolates could be attributed due to the production of antimicrobial compounds. Based on the results, E. coli and Salmonella typhimurium were the most sensitive and S. aureus was the most resistant indicator bacteria against the tested isolates. Table 3. Antibacterial activity of isolates (means ± SD).

Cholesterol Reduction Assay
The removal of cholesterol by 8 probiotics bacterial strains grown on MRS broth supplemented with 0.3% oxgal was presented in Figure 4. All the tested isolates were able to assimilate cholesterol. Results revealed that cholesterol removal varied significantly (p < 0.05) among all the isolates at the same incubation time i.e., 6 h, 12 h and 24 h. Probiotic strains B-2, B-5, B-11 and B-21 showed significantly (p < 0.05) high cholesterol removal (60%, 54%, 71% and 51%, respectively) after 24 h of incubation as compare to other isolates. The lowest cholesterol removal ability was observed in isolate B-28 (38%) and B-33 (33%). The cholesterol removal ability increases as the incubation time increases. Where, no inhibition diameter equal to 0 mm; weak inhibition, diameter between 0-3 mm; good inhibition, diameter between 3-6 mm; strong inhibition, greater than 6 mm.

Cholesterol Reduction Assay
The removal of cholesterol by 8 probiotics bacterial strains grown on MRS broth supplemented with 0.3% oxgal was presented in Figure 4. All the tested isolates were able to assimilate cholesterol. Results revealed that cholesterol removal varied significantly (p < 0.05) among all the isolates at the same incubation time i.e., 6 h, 12 h and 24 h. Probiotic strains B-2, B-5, B-11 and B-21 showed significantly (p < 0.05) high cholesterol removal (60%, 54%, 71% and 51%, respectively) after 24 h of incubation as compare to other isolates. The lowest cholesterol removal ability was observed in isolate B-28 (38%) and B-33 (33%). The cholesterol removal ability increases as the incubation time increases.

Antibiotics Susceptibility
The antibiotic susceptibility of the tested isolates was documented by using different commonly used antibiotics. Results presented in Table 4 revealed that all the isolates were resistant to nalidixic acid, fusidic acid, polymyxin B, neomycin, streptomycin, gentamicin, rifampicin and kanamycin except B-14 which exhibited sensitivity to neomycin, B-21 was sensitive to rifampicin and streptomycin, respectively.

Antibiotics Susceptibility
The antibiotic susceptibility of the tested isolates was documented by using different commonly used antibiotics. Results presented in Table 4 revealed that all the isolates were resistant to nalidixic acid, fusidic acid, polymyxin B, neomycin, streptomycin, gentamicin, rifampicin and kanamycin except B-14 which exhibited sensitivity to neomycin, B-21 was sensitive to rifampicin and streptomycin, respectively.

Hemolytic Activity
Hemolytic activities of 8 tested isolated were evaluated on blood agar plates. None of the tested strains showed α-hemolytic and ß-hemolytic activity when grown on Columbia blood agar plates. The tested strains showed γ hemolytic, i.e., negative, or no hemolytic activity.

Cytotoxicity
In this experiment, MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay was used to observe the effect of different isolates on Caco-2 cell viability. The results showed that all the tested isolates did not significantly damage cells after 8 h of incubation however, 24 h of incubation harmed the cells and cells lost their viability. There was no significant difference in cell viability of B-11 after 24 h of incubation ( Figure 5). Furthermore, least damage was observed in the case of B-14, B-19 and B-21 after 24 h of incubation. Therefore, the probiotic treatment showed insignificant changes among all the tested isolates in the culture supernatant of Caco-2 cells, keeping the membrane integrity during probiotic exposure after 8 h but the viability decreases significantly between 8 to 24 h. Where, inhibition zone diameter <8 mm, resistant (R); inhibition zone diameter between 8-10 mm, moderate (M) and inhibition zone diameter >10 mm, susceptibility (S).

Hemolytic Activity
Hemolytic activities of 8 tested isolated were evaluated on blood agar plates. None of the tested strains showed α-hemolytic and ß-hemolytic activity when grown on Columbia blood agar plates. The tested strains showed γ hemolytic, i.e., negative, or no hemolytic activity.

Cytotoxicity
In this experiment, MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay was used to observe the effect of different isolates on Caco-2 cell viability. The results showed that all the tested isolates did not significantly damage cells after 8 h of incubation however, 24 h of incubation harmed the cells and cells lost their viability. There was no significant difference in cell viability of B-11 after 24 h of incubation ( Figure 5). Furthermore, least damage was observed in the case of B-14, B-19 and B-21 after 24 h of incubation. Therefore, the probiotic treatment showed insignificant changes among all the tested isolates in the culture supernatant of Caco-2 cells, keeping the membrane integrity during probiotic exposure after 8 h but the viability decreases significantly between 8 to 24 h.

Molecular Identification of Isolate (B-11)
Considering that among all the isolates, B-11 possessed most of the probiotic's attributes. The isolate was further confirmed through 16S rRNA gene sequence analysis. On the basis of molecular identification, purified PCR products were sequenced and compared with the information in the NCBI database. BLAST search disclosed that isolated bacterium as B. longum subsp. longum which

Molecular Identification of Isolate (B-11)
Considering that among all the isolates, B-11 possessed most of the probiotic's attributes. The isolate was further confirmed through 16S rRNA gene sequence analysis. On the basis of molecular identification, purified PCR products were sequenced and compared with the information in the NCBI database. BLAST search disclosed that isolated bacterium as B. longum subsp. longum which exhibited a similarity index (100%) to other B. longum strains. Therefore, the isolate was a strain of B. longum and designated as B. longum B-11. The 16S rRNA sequence was deposited to NCBI Genebank data base under the accession number MH041649.1.

Scanning Electron Microscopy of Rat Intestine
Scanning electron microscopy images revealed that small intestine of rat (control group, G 0 ) showed a large number of coccus shaped bacterial attachment on the upper surface (Figure 6a), while the rats treated with B. longum B-11 (G 1 ) showed presence of a large number of rods shaped bacteria (B. longum B-11). Well organized and steady microvilli can be observed in the case of B. longum B-11 treated rats (Figure 6b,c).

Scanning Electron Microscopy of Rat Intestine
Scanning electron microscopy images revealed that small intestine of rat (control group, G0) showed a large number of coccus shaped bacterial attachment on the upper surface (Figure 6 a), while the rats treated with B. longum B-11 (G1) showed presence of a large number of rods shaped bacteria (B. longum B-11). Well organized and steady microvilli can be observed in the case of B. longum B-11 treated rats (Figure 6 b,c).
The probiotic potential of isolates was assessed through their survival ability under the artificial simulated conditions in the digestive tract. Various researchers contributed their work in this field to evaluate potentially probiotic strains. pH is an important parameter that affects the growth and viability of probiotics during gastric transit. Among eight isolates, three isolates (B-2, B-11, B-19) showed survival rate in the range of (75-85%) at pH 2 and more than 90% at pH 3 after 2 h of exposure. The survival rate is higher as compared to previously reported Bifidobacterium strains under such low pH [42,43].
Probiotics should survive in the gastrointestinal conditions and reach alive in the small intestine, where they colonize and impart positive health benefits to the host. The decreased in the viability and survival is due to the presence of pepsin enzymes and low pH of gastric juice [44]. The probiotics should with stand harsh gastrointestinal conditions (gastric juice and bile salt) to exert positive health benefits to humans. Gastric juice and bile salt-resistance is considering one of the prime criteria in the selection of probiotics [45]. B. longum strains presented maximum viability in GIT as compared to other species belonging to the same genera [42].
All the isolates exhibited more than 70% survival rate at 0.1% (w/v) bile salt concentration except B-19. Three isolates (B-2, B-11, B-28) showed a survival rate in the range of (70-75%) at such a high concentration of bile salt (1%). Sanchez, et al. [46] studied the probiotic potential of different B. longum strains (mutant and wild type) at different bile salt concentrations (0.5%, 1%, 2% and 3%) and expounded that viability of B. longum decreased as bile salt concentration increased. Mutant strains indicated viability at 0.5% to 3% bile salt concentration while the wild type was not able to grow at such a high concentration of bile salt. Haros, et al. [47] investigated tolerance of B. pseudocatenulatum to (0.5-2%) bile salt and reported that Bifidobacterium strain was resistant to bile salt even after 4 h of incubation. Ren, Li, Qin, Yin, Du, Ye, Liu, Liu, Wang, Li, Sun, Li, Tian and Jin [10] demarcated similar results for bile salt tolerance at 0.3% to 0.5% concentration. Even most of the probiotic strains showed resistance to 1% bile salt, which is approximately three times the bile concentration in the human intestine. Haghshenas, et al. [48] observed tolerance to bile salt but survival decreased as a function of incubation time. In the current study, all isolates have the ability to tolerate high phenol concentration (0.4%), revealed that they can tolerate the bacteriostatic effect of phenol in GIT. Phenol is actually the by-product of metabolism of aromatic amino acids that produce in the gut.
Exopolysaccharide production is important for protection, colonization and acts as intermediaries to establish an association between bacteria and host [49]. All the isolates were able to produce EPS. Among Bifidobacterium strains, B. longum is the most frequently cited probiotics due to the comparatively high yield of EPS being produced. Prasanna, et al. [50] screened various Bifidobacterium strains and reported that among all the Bifidobacterium strains, B. longum subsp. infantis CCUG 52,486 was the highest EPS producing strain (138 mg/L). The capsular structure of EPS protects probiotics from harsh gastrointestinal conditions like gastric juice, bile salt and etc. High yielding EPS strains could survive well in high acidic and bile salt conditions. In this way, EPS promotes bacterial growth and survival in the presence of high acid and bile salt conditions [51]. EPS also serves as a growth substrate for gut microflora and provides protection against phagocytosis and bacteriophage attack.
Cell surface properties i.e., cell surface hydrophobicity and autoaggregation are important for bacterial attachment to intestinal lining and colonization in the GIT. In the current study, B-5 (74.21%), B-11 (78.89%) and B-28 (71.99%) exhibited comparatively high hydrophobicity to xylene. These isolates also showed high autoaggregation B-5 (55%) B-11 (65%) and B-28 (58%) after 24 h of incubation. Xu, Jeong, Lee and Ahn [28] described cell surface property and reported that the highest value for hydrophobicity was found in B. longum B6 (53.6%) followed by Lb. rhamnosus GG (46.5%). Moreover, Rahman, et al. [52], also explored 13 strains of 4 different Bifidobacterium spp. for their auto-aggregation ability and surface hydrophobicity. They explained that among all Bifidobacterium spp. B. longum presented high cell surface hydrophobicity. Auto-aggregation ability and surface hydrophobicity are related to cell adhesion, which promotes binding of probiotics to the intestinal lining. As a result of this binding, it acts as a barrier and pathogens are unable to colonize. This adhesion of probiotics to the intestinal cell is essential for pathogen omission and immunomodulation [53]. In vitro, cell surface properties are not a reliable approach to judge their interaction within the host cell. There is still a need to study it in vivo, for a better understanding of this interaction.
Bifidobacterium has the ability to act as an antioxidant by quenching free oxygen radicles. In the current study the cell-free supernatant of B-2 (80.29%), B-5 (84.44%), B-11 (87.72%), B-28 (81.19%), B-33 (80.72%) exhibited strong antioxidant activity as compared to other studies. Shen, et al. [54] documented the antioxidant activity of B. animals 01 both in vitro and in vivo. The results reported that culture supernatant exhibited the highest DPPH radical scavenging activity (73.11%). Likewise, Wang, et al. [55] elucidated that, LAB and Bifidobacterium fermented soymilk had higher antioxidative activity than un-fermented soymilk. Nevertheless, these studies represented the antioxidative ability in vitro but there is still a need to study this activity in vivo. Synthetic antioxidants have serious health implications regarding their safety and long term utilization. Synthetic antioxidants i.e., butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA) have toxic and carcinogenic effects [56]. Therefore, it is the need of the day to explore non-toxic, natural and low-cost antioxidants as a substitute for a synthetic antioxidants in pharmaceutical and food industries.
Nitrite is commonly used as a food additive and has been widely found in various food products. It is an important N-nitrosamines precursor, which potentially causes cancer and methemoglobinemia. Nitrites also react with amines (protein degradation products) to form N-nitroso compounds [10]. So, it is an important safety concern to limit the use of nitrite in food products. In this context, all the tested isolates have the ability to deplete nitrite and restrict the conversion of nitrite into nitrosamines. Although the exact mechanism is still not fully understood it might be due to enzymatic and chemical reactions. There is a need to further evaluate this in a food product.
Probiotics are helpful in the management of GIT infections. They exert positive effects against antibiotic-associated diarrhea, travel diarrhea and rotavirus. These antimicrobial substances are proteinases in nature and produced by various Bifidobacterium spp. Collado, et al. [57] explicated that antimicrobial peptides produced from Bifidobacterium strains had strong antagonist activity against both antibiotic sensitive and antibiotic-resistant pathogens. Antimicrobial peptides could be one of the effective mechanisms to combat infectious diseases.
An isolate B-11 exhibited most of the probiotics attributes and confirm through molecular identification and named as Bifidobacterium longum B-11. The obtained sequence was deposited into NCBI GenBank and allotted NCBI accession number (MH041649.1). B. longum B-11 was further selected for in vivo attachment to the intestinal lining and the results are in line with the findings of Choudhary, Dubey, Sengar and Dheeman [40]. B. longum B-11 was the potential microorganism to be used as probiotics for human consumption due to better colonization in GIT and better adhesion to the epithelial lining of the host.
High cholesterol level has several health implications i.e., high risk of cardiovascular ailments which is the major cause of death. Use of the drug in the treatment of hypercholesterolemia has numerous side effects. Therefore, it is of utmost important to use natural, cost-effective methods to reduce serum cholesterol. Consumers are more concerned to use safe and alternate products for hypercholesterolemia that limit dependence on drug therapy. In this context, the use of supplementing diets with probiotic strains is one of the effective and promising strategies to reduce serum cholesterol. The results of the current study revealed that Bifidobacterium strains have the ability to lower down the cholesterol level. B. longum B-11 exhibited maximum cholesterol removal percentage. The result of the current study is in accordance with the findings of El-Gawad, et al. [58], who evaluated that the cholesterol reduction of B. longum Bb-46 fortified buffalo milk yogurts. They fed hypercholesterolemic male albino rats with 50 g yoghurt containing 0.07% (w/v) probiotics for 35 consecutive days. They delineated that probiotic fortified yoghurt significantly reduced total cholesterol level by 50.3%, triglycerides by 51.2% and LDL by 56.3% as compared to control. Likewise, Xiao, et al. [59] explicated the effect of low fat yoghurt fortified with B. longum BL1 (10 8 CFU/g) on cholesterol level of thirty-two subjects (cholesterol level 220-280 mg/dL, aged 28-60 years old, weight 55.4-81.8 kg). The results illustrated that there was a significant reduction in serum total cholesterol level, LDL cholesterol and triglycerides as compared to control after 4 weeks.
As far as safety is concerned the isolates should have the ability to resist commonly used antibiotics [10]. The literature revealed that Bifidobacterium is susceptible to antibiotics and can be consumed safely after antibiotic therapy for maintaining the balance of gut microflora. Similarly, Zhou, et al. [60] investigated that B. lactis strains were susceptible to erythromycin, novobiocin, rifampicin, tetracycline, spectinomycin, h-lactam, chloramphenicol, penicillin, cephalothin and ampicillin. These strains were resistant towards nalidixic acid, fusidic acid, polymyxin B, aminoglycosides, neomycin, streptomycin, gentamicin and kanamycin. Likewise, Temmerman, et al. [61] isolated B. longum from different products and evaluated that B. longum was resistant to kanamycin and susceptible to erythromycin, tetracycline, chloramphenicol, penicillin and vancomycin.
According to the European Food Safety Authority (EFSA), the evaluation of hemolytic activity is strongly recommended if the isolated bacteria intended to use in food products, even if they have GRAS or QPS (Quality Presumption of Safety) status (FAO/WHO 2006). In this study, the hemolytic activity of 8 tested isolates was evaluated on Columbia blood agar plates. None of the tested strains showed αhemolytic and β-hemolytic activity when grown in Columbian sheep blood agar. All the tested strains showed γhemolytic or no hemolytic activity. Our results were in agreement with the finding of Oh and Jung [62] who determined the hemolytic activity of five Lactobacillus species isolated from traditionally fermented millet-based alcoholic beverages. The results of current findings were also consistent with the results of Wang, et al. [63] who evaluated the probiotic potential of lactic acid bacteria from Chinese spontaneously fermented non-dairy food products and revealed no hemolytic activity of probiotic. Many researchers [37,[64][65][66] reported that probiotics did not showed hemolytic activity. When the safety of probiotic is concerned, lack of hemolytic activity is important during the selection of probiotic strains, because such strains are non-virulence and lack of hemolysin ensures that virulence will not appear among the bacterial strains (FAO/WHO 2006). The enzymes production that are capable of degrading mucin is suggested as a key factor of virulence for pathogens. So that, this feature is not recommended for probiotic strains because it modifies the intestinal mucosal lining as a result mucosal invasion happens by other toxic materials and pathogens [37].
It is proved that all the tested isolates were found to have better anticancer activity with cell viability > 80% of the Caco-2 cell. Thus, the novelty of isolates especially B-11 has been proved for its effective anti-carcinogenic effect. Chui and coworkers reported similar results, L. plantarum co-cultured with Caco-2 cells did not affect cell viability within 10 h, but cell viability decreased significantly between 12 and 14 h but the decreases was non-significant [67]. The inhibitory effects of probiotics upon the proliferation of several colon cancer cell lines have been previously demonstrated by various authors [38,[68][69][70][71][72][73].
MTT assay is considered as a highly sensitive assay for determining cellular respiration, cell viability and cytotoxicity because only living cells are able to produce formazan products during this reaction. Lactobacilli and bifidobacteria are the most prominent probiotics that have been recognized as safe and increasing research interest due to their significant role in the prevention of cancer [74]. Probiotics generally modulate intestinal microflora, inactivate carcinogenic complexes, improves host's immunity, compete with pathogens, have anti-proliferative effects by regulating apoptosis and cell differentiation [68].

Conclusions
In the current study, eight potential probiotics were successfully isolated from raw camel milk according to the international probiotic guidelines. Among all the isolates, B-11 had the maximum potential for practical application due to high resistance to GIT conditions as well as strong antagonistic and antioxidant activities. B-11 also displayed antibiotic susceptibility and produced the highest EPS yield. B-11 is effective to deplete nitrite and assimilate cholesterol. Therefore, B-11 is introduced as a potential probiotic to be used safely in the development of functional food products. Moreover, in vivo studies would be needed to further evaluate its immunomodulatory, safety and health perspective as a potential probiotic.