Functional characteristics of Lactobacillus strains isolated from camel's milk.

This study aimed at isolation, identification and evaluation of probiotic potential of Lactobacillus isolates from camel's milk. Thirty four Lactobacillus isolates coded M 1 to M 34 were Gram positive, rods, catalase and oxidase negative and nonspore-forming bacteria. These isolates were identified by biochemical tests and API 50 CH kits. From these, 14 different Lactobacillus isolates (M 1, M 2, M 4, M 5, M 9, M 10, M 12, M 14, M 15, M 18, M 20, M 27, M 29 and M 31) which were tolerant to gastric and intestinal juices in a previous study were now tested for antipathogenic activity which varied according to the Lactobacillus species and the challenged pathogen. All 14 isolates demonstrated significant inhibitory effect against methicillin resistant Staphylococcus aureus (MRSA), Bacillus cereus and moderate to low activity against Salmonella typhimurium and Escherichia coli. When tested for bile tolerance at the concentration of 0.3 to 2.0%, the growth rate of 8 isolates M 2, M 5, M 9, M 10, M 12, M 14, M 18 and M 20 exceeded 60% in 0.3 and 0.5% bile. Original Research Article Abbas and Mahasneh; BJMMR, 7(1): 25-39, 2015; Article no.BJMMR.2015.304 26 M 2 (L. fermentum) and M 12 (L. plantarum) and M 20 (L. paracasei ssp. paracasei) exhibited the highest growth rates of 82, 79.4 and 78.8% respectively. At higher levels of 1 and 2% bile, significant reduction (p < 0.05) was observed for all tested isolates except M 9 (L. plantarum) with growth rate of 66.5% at 2% bile. As for cholesterol reduction, M 10 (L. plantarum) and M 15 (L. paracasei ssp. paracasei) had the highest reduction rate of 58.0 and 53.2% respectively, which is comparable to the reference strain L. reuteri DSMZ 20056. Testing adhesion to intestinal epithelial cells and ileal tissues of BALB/c mouse; M 20 (L. paracasei ssp. paracasei) and M 2 (L. fermentum) exhibited highest attachment rate of more than 15 bacterial cells/epithelial cell. SEM images showed variable degrees of bacterial attachment to ileal tissues. These results suggest that camel milk is a rich source for potential probiotic lactobacilli which may be suitable for food and nutraceuticals industries; however, further in vivo investigations are needed.


INTRODUCTION
Probiotics refer to live, non-pathogenic microbial preparations that beneficially exert health benefits on the host when administered in adequate amounts [1,2]. Estimates claim that 500-1000 different bacterial species are present in the human intestinal tract [3]. This involves both pathogenic and non-pathogenic microorganisms which are present in a varying complex symbiosis over human life-span [4]. Lactobacilli group are the first residents in the gastrointestinal tract after birth [5]. In healthy persons, they are present in the oral cavity, ileum, colon and in the vagina [6]. Lactobacilli is a group of Gram-positive, non-spore forming, catalase negative rods producing lactic acid by homo-or heterofermentative activity of carbohydrates [7][8][9][10]. Lactobacilli have been long known as the main microbiota in dairy industries [11]. Fermentation and other products of lactobacilli are extremely beneficial in controlling pathogens thus preventing spoilage of fermented foods among other functions [12].
To exert health benefits, live probiotic bacterial counts should be at the level of 10 7 cfu/ml of the product at the time of consumption [12]. Previous studies indicated healthy functions of lactobacilli properties pertaining to modulation of gut flora, reduction of gastrointestinal syndromes, diarrhea reduction, immune system enhancement, antimutagenic and anticarcinogenic activity and cholesterol lowering effects [7,13,14]. To achieve some or all of these suggested activities, probiotic microorganism should exhibit characteristics to overcome extreme low pH and bile toxicity, adherence to human intestinal mucosa, inhibitory activity against pathogens and production of antibacterial substances [15][16][17]. The continued search for new and novel probiotic bacterial strains is indispensable to obtain new functional products and also important to reach a state of producing more active probiotic cultures and hopefully producing designer selective probiotics for specific purposes. The quest in this direction, lead researchers to mine natural resources specially traditionally fermented foods for unique lactobacilli [18][19][20][21].
In this study, camel's milk both fresh and spontaneously fermented products which rarely been studied were used to isolate and identify potential probiotic strains of Lactobacillus and to study their bile tolerance, adhesion to intestinal tissues, cholesterol reducing level and antimicrobial activity against selected human pathogenic bacteria. These characteristics are among criteria of potential probiotics, hence are tested in this study. Results obtained may also help in clarifying the common belief in this region, of the curing ability of camel's milk products for several ailments [22,23] which may be linked to the presence of such probiotic bacteria.

Collection of Milk Samples and Enrichment for Indigenous Bacteria Growth
Ten samples (500 ml each) of raw camel's milk were collected from dromedary camel herds from different sites of Jordan during the period extending from April 2009 -May 2010. The samples were collected by manual milking in sterile plastic bottles, kept on ice and transported to the microbiology laboratory of the University of Jordan within 2 h of collection. Aliquots of the samples were used directly and the remainder was allowed to ferment spontaneously through the raw milk indigenous microorganisms at room temperature, without any additives. The enrichment process of the collected samples was achieved by adding 10 ml of raw camel milk to 80 ml MRS broth medium (Oxoid, UK). The enriched samples were incubated at 30ºC and/or 37ºC for 1 week under static and/or shaking conditions.

Isolation of Bacterial Strains and Culture Conditions
Lactobacillus species were isolated from camel's milk by aseptic microbiological procedures. Briefly, selective medium for Lactobacillus de Man Rogosa and Sharpe (MRS) agar plates were used. One hundred microliters of tenfold dilution of milk samples in sterile normal saline were spread on the surface of MRS agar plates. Incubation was carried out by incubating lots at 30ºC and others at 37ºC. After 2-5 days incubation at anaerobic conditions using anaerogen sachet (AnaeroGen, UK) in anaerobic jar (Oxoid, UK), suspected Lactobacillus colonies were picked off and subcultured onto the same medium. In some cases, MRS agar supplemented with bromocresol purple (0.01% w/v) to obviate colonies of Lactobacillus species was used. To enhance isolation, MRS medium was supplemented with 0.5 g / L cysteine-HCl.

Maintenance of Isolated Strains
Isolated strains were stocked as frozen cultures in MRS broth with 20% glycerol at both -80ºC and -20ºC. Working cultures were kept on MRS agar slants and MRS agar plates at 4ºC and were routinely subcultured every 4 weeks.

Biochemical Identification of Bacterial Strains
All isolates were tested for catalase and oxidase activity, Gram reaction, cell morphology and spore formation. All Gram positive and catalase negative rods were tested for growth in MRS broths at 10, 37 and 45ºC and for growth at pH 3.9 and 9.6. The strains were tested for production of acids from carbohydrates and related compounds by using API 50 CH kits and CHL media ((BioMérieux, France). The API test strips were prepared according to manufacturer's instructions. Results were scored after incubation for 24 and 48 h at 37ºC. These results were joined to the apiweb TM identification software with database (V5.1), which uses the phenotypic data to predict a species identity. Interpretations of the fermentation profiles were facilitated by analytically comparing all results obtained for the isolates studied with information from the computer-aided database.

Bile Tolerance Test
The tolerance of the bacterial isolates to bile was tested using MRS broth prepared with and /or without 0.3, 0.5, 1 and 2% (w/v) oxgall (Oxoid, UK). Ten milliliter aliquots of bile solutions were transferred into standard glass tubes and sterilized by autoclaving at 121ºC for 15 min. For each new bacterial culture to be tested, three tubes of each concentration were inoculated with 0.2 ml of freshly prepared MRS broth culture of 0.5 McFarland. Inoculated tubes were incubated at 37ºC for 24 h. Bacterial growth was recorded by measuring optical density at 600 nm using a spectrophotometer (Biotech, UK). Growth ability (bile tolerance) was expressed as a percentage of that of the control (inoculated tubes of MRS broth without oxgall), which was assigned a value of 100%.

Antipathogen
Activity of Lactobacillus Isolates

Agar spot method
The antibacterial activity of the selected Lactobacillus isolates was determined by the agar spot test described by Schillinger and Lücke (1989) [24] with some modifications as follows: five microliters of each overnight culture of Lactobacillus isolate were spotted onto the surface of MRS agar plates (containing 0.2% glucose) and were then incubated under anaerobic conditions at 37ºC for 48 h. An overnight culture of four indicator strains E. coli ATCC 25922, S. typhimurium ATCC 14028, B. cereus (Toxigenic strain, TS), and methicillin resistant S. aureus (MRSA clinical isolate) were grown in nutrient broth and were adjusted to 0.5 McFarland and then were diluted 1:10 using nutrient broth to reach 10 7 CFU/ml. Aliquots of 0.25 ml were inoculated into 7 ml of soft nutrient agar (containing 0.2% glucose and 0.7% agar). Inoculated soft agar was immediately poured in duplicates over the MRS plate on which the tested Lactobacillus isolate was grown. The plates were incubated aerobically at 37ºC for 24 h.
The antibacterial activity was detected by measuring the diameter of inhibition zones around the Lactobacillus bacterial spots. Inhibition was recorded as positive if the diameter of the zone around the colonies of the producer was 2 mm or larger [25].

Cholesterol-lowering Effect
To test Lactobacillus for cholesterol-lowering effect, one fresh colony from each Lactobacillus isolate was inoculated into 5 ml MRS broth separately and incubated anaerobically for 24 h at 37ºC. Then, they were inoculated (1%) into MRS-THIO broth with 0.1 g/L filter-sterilized water-soluble cholesterol (polyoxyethanyl cholesteryl sebacate) (Sigma-Aldrich, USA) and incubated under anaerobic conditions at 37ºC for 24 h. After the incubation period, cells were centrifuged at 12,000 x g at 4ºC for 10 min, and the remaining cholesterol concentration in the broth was determined using o-phthalaldehyde modified colorimetric method as described by Rudel and Morris (1973) [26]. One milliliter of the supernatant (broth containing the remaining cholesterol) aliquot was added with 1 ml of KOH (50% w/v) and 2 ml of absolute ethanol, vortexed for 1 min, followed by heating at 37ºC for 15 min. After cooling, 2 ml of distilled water and 5 ml of hexane were added and vortexed for 1 min.
The hexane layer of 2.5 ml was transferred into a glass tube. The hexane was evaporated from each tube at 60ºC under the flow of nitrogen gas. The residue was immediately dissolved in 2 ml of o-phthaldehyde reagent. The reagent contained 0.5 mg of o-phthaldehyde per ml of glacial acetic acid. After complete mixing, the tubes were allowed to stand at room temperature for 10 min, and then 0.5 ml of concentrated sulfuric acid was added and the mixture was vortexed for 1 min. After standing at room temperature for an additional 10 min, the absorbance was read at 550 nm against a reagent blank. The removal rate of every strain was computed by the following formula: the cholesterol reducing rate = [(A 0 -A) / A 0 ] × 100%. Where, A 0 : absorbance of the unfermented broth. A: absorbance of the broth fermented for 24 h.

Mouse epithelial cells preparation
BALB/c mouse was sacrificed using high dose of ether and segments of the ileum were taken and opened, washed with phosphate buffer saline (PBS) (pH 7.2) and held in 10 ml PBS at 4ºC for 30 min to loosen the surface mucus. The segments were then rinsed thrice with PBS and the epithelial cells were scraped off with the edge of a microscopic slide and were suspended in PBS. The indigenous bacteria were removed and eliminated completely by washing the cells suspension three times with 10 ml of PBS and centrifugating at 100 x g at 2ºC, and then were examined microscopically to ensure that the adherent bacteria were removed.

Lactobacillus cells preparation
Selected Lactobacillus species were grown in MRS broth overnight at 37 °C. Aliquots of these were centrifuged and resuspended in PBS to give a cell density of 1×10 8 CFU/ml.

Adhesion assay
Five hundred microliters of each bacterial suspension were added to 500 µl of epithelial cell suspension separately and the mixtures were rotated at 35 rev/min at 37ºC for 1 hour. Then the non-adherent bacteria were removed by centrifuging the mixture for 10 min at 100 x g. The supernatants were discarded and the pellets were resuspended in 1 ml PBS and were washed thrice under the same conditions. The bacterial binding to the epithelial cells in the pellet was measured by observing Gram stained preparations with light compound microscope (Novex, Holland). Positive adhesion was recorded if more than 15 bacterial cells adhered to one epithelial cell [27]. BALB/c mouse was sacrificed using high dose of ether and segments of the ileum were taken and opened, washed with phosphate buffer saline (PBS) (pH 7.2) and were held in 10 ml PBS at 4 °C for 30 min to loosen the surface mucus. The segments were then rinsed thrice with PBS. The indigenous bacteria were removed and eliminated completely by washing the ileal segments three times with 10 ml of PBS and centrifuging at 100 x g at 2ºC then the samples were examined microscopically to ensure that the adherent bacteria were removed.

Lactobacillus cells preparation
Selected Lactobacillus species isolates were grown in MRS broth overnight at 37ºC and were then centrifuged and resuspended in PBS to give a cell density of 1 × 10 8 CFU/ml.

Adhesion assay
One milliliter of each bacterial suspension was added to one ileal segment separately and the mixtures were rotated at 35 rev/min at 37ºC for 1 h. Then the non-adherent bacteria were removed by centrifuging the mixture for 10 min at 100 x g. The supernatants were discarded and the pelleted tissues were resuspended in 1 ml PBS and washed thrice under the same conditions. The bacterial binding to the ileal tissues in the pellet was observed by scanning electron microscopy (Inspect F 50, Netherlands).

Specimen preparation for scanning electron microscopy
Pellets of the ileal tissues binding bacteria were fixed with 1.5% glutaraldehyde solution (25% reagent) prepared in 0.1 M PBS (pH 7.2) and incubated at 4ºC overnight. After the fixation step, specimens were rinsed first with 0.1 M PBS for 10 min, and then rinsed further three times for 20 min each at 4ºC in order to remove the excess fixative. The dehydration was performed with a graded series of ethanol. Specimens were immersed in 30% ethanol for 2 min, 50% ethanol for 5 min, 70% ethanol for 10 min, 90% ethanol for 15 min, and finally 100% ethanol twice for 20 min at 4ºC. Ready specimens were then mounted on a holder that can be inserted into the scanning electron microscope. They were mounted on aluminium stubs using a doublesticky tape. Specimens were coated with a thin layer of approximately 20 nm to 30 nm of conductive metal, platinum, using a sputter coater (Bruker, Germany).

Statistical Analysis
The results are presented as means ± S.D. Statistical differences among bacterial isolates in the in vitro study were determined by two way ANOVA except for cholesterol reduction experiment which was determined by one way ANOVA. Differences were considered significant at p < 0.05.

Lactobacillus Isolation
Thirty four confirmed Lactobacillus isolates coded M 1 to M 34 were isolated and identified using biochemical and API 50 CH kits. These isolates were diverse in terms of identity. However, the majority belonged to L. fermentum, L. plantarum and L. paracasei ssp. paracasei. As scarce as it is the case with studies on micro flora of camel's milk, Yateem et al. [28] reported the isolation of some lactobacilli from camel milk samples from Kuwait. It was also observed that some isolates of the same species varied slightly, an observation reported by Suriasih et al. [29] with Lactobacillus isolates.
Recently, Akhmetsadykova et al. [30] studied the flora of camel milk and among others; they were able to isolate lactobacillus species.  (Table 1). Siezen et al. [31] reported variations on phenotypic and genotypic levels within the genus Lactobacillus specially with isolates of different environments. Experimental results of this study (Table 1) showed that Lactobacillus species from camel milk were mostly able to survive acidic pH (3.9) and the extreme alkaline pH of 9.6, unlike results of Ammor et al. [32] where little of their isolates tolerated such pH values. Ashmaig et al. [33] results of Lactobacillus isolates from camel milk showed somehow a similar trend in response to different parameters.   (Table 3). At 1% and 2% bile, M 2 (L. fermentum) and M 9, M 10 and M 12 which belong to L. plantarum showed relative growth rates above 65% and above the L. reuteri DSMZ 20056 which is a reference probiotic strain [39]. Most of the other isolates were significantly lower (p < 0.05) than these values and comparable with the reference strain. Bile is fundamental in the defenses of the gut [40]. The range of physiological concentrations of the human bile lies between 0.3 to 0.5% [41]. As presented above considerable species and strain variations to bile resistance are observed in this study and other studies [42], most likely due to expression of bile resistance mediator proteins by bacterial cells [43]. Some of our isolates specially L. plantarum isolates M 9, M 10 and M 12 (Table 3) were superior to lactobacilli isolates of Kaboré et al. [44] and comparable to Tambekar and Bhutada [45] isolates.

Bile Tolerance
These variations in bile tolerance of isolates of this study agrees with previous findings of probiotic cultures being species as well as strain specific in response to bile concentrations [46,47].
Bacterial salt tolerance is indirectly related to cholesterol lowering through its incorporation into the cellular membranes of probiotic bacteria from the media during growth [48,49]. Current research findings suggest that probiotic bacterial function in the detoxification of bile salts increases their intestinal survival and persistence of producer strains [50,51]. This in turn improves the efficiency of the probiotic strains [52] and forms an important basic property in screening for novel probiotic strains. However, sensitivity to bile salts may be related to absence of bile salt hydrolase among other factors [12].

Cholesterol Lowering Effects
All selected Lactobacillus isolates (Fig. 1) showed high ability of cholesterol in vitro reduction. No significant (p < 0.05) variations were recorded. However M 10 (L. plantarum) was superior (58% reduction rate) to others as well as to the control strain of L. reuteri DSMZ 20056 (53.2%) which was similar to M 15 (L. paracasei ssp. paracasei) (53.2%). M 31 (L. rhamnosus) had the lowest reduction rate of 41%. Zheng et al. [40] isolated probiotic Lactobacillus strains including L. plantarum B23 which were able to assimilate and precipitate cholesterol with variations between isolates of the same and different species. It is recognized that high cholesterol levels in the human blood is a risk factor for coronary heart diseases [53]. It is also known that Lactobacillus strains that were able to assimilate cholesterol in vitro were also capable of reducing it in vivo [54,55]. In this study, the 14 selected Lactobacillus strains showed a well defined potential for cholesterol reduction in vitro. Some of these isolates as indicated above were superior even for L. reuteri DSMZ 20056 which is known in probiotic understanding to be of great cholesterol lowering effect [56]. These results agree with other findings Wang, et al. [41] both in vivo and in vitro. It is now known that lactobacilli with lowering cholesterol activities do that in multiple ways [57][58][59]41]. Lavanya, et al. [60] isolated lactic acid bacteria from fermented milk which assimilated and reduced cholesterol levels at the rate of 28-83%. Isolates of this study needs further testing to substantiate their probiotic characteristics in vivo as well as on improving their technological properties.    (Fig. 3C). Adhesion and colonization of probiotic bacteria is an essential character to express their health benefits [27,61]. It is established now that adhesion is a prerequisite for colonization and antagonistic activity against enteropathogens and immunomodulations [62,63]. Isolates of this study exhibited attachment ability to both epithelial cells and ileum tissue of BALB/c mouse and they were significantly better adherent than the reference probiotic strain of L. reuteri DSMZ 20056. Tsai et al. [64] found that Lactobacillus strains of animal origin were able to adhere strongly to different types of epithelial cells isolated from BALB/c mouse. Both et al. [65] recorded good adhesion ability of L. acidophilus and L. casei to epithelial cells. Martín et al. [66] attributed the good adhesion capacity of lactobacilli to mucin mediated by an extracellular form of glyceraldehyde 3phosphate dehydrogenase. Although the results of in vitro testing may not be the same as in vivo [67], we can be sure that an association between adhesion ability and health benefits of probiotic bacteria exists [27,66]. It is concluded that the adhesion ability of lactobacilli is relatively dependent on variations among species, strains and their origin. This is also linked to adhesion factors on bacterial cell surfaces which invites for further investigations.
Finally, looking at the above results, it is rather clear that camel's milk is a good and unique source of interesting probiotic Lactobacillus strains. Most isolates exhibited significant activities pertaining to criteria needed for any bacterial isolate to be of potential use as a probitic in foods industries. Additionally, this study on camel milk shed some light on the traditional belief in Middle-Eastern countries that camel milk and products have a curative abilities as a nutraceutical against several ailments [68,23], a claim which needs further in vivo studies.

CONCLUSION
This study proved that camel milk may be used to isolate unique probiotic lactobacilli isolates. These isolates met most criteria needed for a potential probiotic bacterial isolate. Results also may substantiate the belief in the curative abilities and probable use of camel milk as a nutraceutical food product.

CONSENT
Not applicable.