Characterization of Probiotic Strains of Lactobacillus Candidates for Development of Synbiotic Product for Kazakh Population

1Head of Laboratory of Microbiology in RSE "Republican Collection of Microorganisms" SC MES RK (Astana, Kazakhstan). 2Senior Researcher of Laboratory of Microbiology in RSE "Republican Collection of Microorganisms" SC MES RK (Astana, Kazakhstan). 3Deputy General Director for Science in RSE "Republican Collection of Microorganisms" SC MES RK (Astana, Kazakhstan). 4Junior researcher of Laboratory of Microbiology in RSE "Republican Collection of Microorganisms" SC MES RK (Astana, Kazakhstan). 5Head of Laboratory of Genetics and Biochemistry of Microorganisms in RSE "Republican Collection of Microorganisms" SC MES RK, (Astana, Kazakhstan). 6Director General in RSE "Republican Collection of Microorganisms " SC MES RK (Astana, Kazakhstan).

The microbial ecology in the gastrointestinal tract influences many functions in our body.These are digestion, absorption of nutrients, detoxification.It is finally affecting the functioning of immune system.Hence, the balance in microbiota of gut is focused to provide the colonization resistance against infectious agents and to promote anti-allergicprocesses and to reduce hypersensitivity 1 .
Probiotics have been defined as viable microorganisms that, when administrated in adequate amounts, exert beneficial effects in the prevention and treatment of specific pathologic conditions.Studies have suggested that they enhance gut barrier function, normalize intestinal milieu, synthesize antibacterial substances, and stimulate immunity [2][3] .Also, probiotics such as these have good safety and tolerability profiles, and side effects are uncommon 4 .
Lactobacilli are an important part of the normal flora commonly found in the mouth, gastrointestinal tract and female genitourinary tract 5 .Because they produce organic acids, hydrogen peroxide and bacteriocins, many strains of lactobacilli show antagonistic activity toward pathogenic and conditionally pathogenic microorganisms.Recently, increasing attention has been given to their probiotic, health-promoting capacities, among which their antagonistic potential against pathogens plays a key role [6][7] .
Probiotic potential included survival in gastrointestinal simulated juice, antagonistic and bacteriocin activity, acid pH and bile tolerance, antibiotic resistance to antibiotics, adhesion ability and others [9][10][11] .
The genera Lactobacillus, Lactococcus, Leuconostoc, Pediococcus, and Enterococcus have been associated with more than 300 different bacteriocins.The inhibition spectra of the bacteriocins produced by these lactic acid bacteria can be broad or narrow, but in general the bacteriocins exhibit inhibition against closely related Gram-positive bacteria, although several bacteriocins have been shown to be active against certain Gram-negative bacteria, including pathogenic species of Escherichia and Salmonella [12][13][14] .Bacteriocins might warrant serious consideration as alternatives to traditional antibiotics.These molecules exhibit significant potency against other bacteria, including antibiotic-resistant strains 15 .Resistance to antimicrobial drugs (antibiotics) is a common characteristic in the world of lactic acid bacteria 16 .Moreover, Lactobacilli, Pediococci and Leuconostoc spp.have been reported to be highly resistant to vancomycin and some Lactobacilli have high resistance to bacitracin, cefoxitin, ciprofloxacin, fusidic acid, streptomycin, sulphadiazine, teicoplanin, vancomycin, erythromycin, gentamicin and kanamycin [17][18][19][20] .Lactic acid bacteria may serve as reservoirs of antibiotic resistance genes potentially transferable to human pathogens.Hence, there is a growing interest in the possible role of lactic acid bacteria as vectors of antibiotic resistance determinants 21 .Tolerance to NaCl, gastric acid and bile has thus become important selection criterion for probiotic strains [22][23] .Different stress factors might considerably affect viability or performance of the Lactobacillus strains 24 .
The objective of this study was to identify the probiotic properties of Lactobacillus strains to develop the synbiotic product, based on Lactobacillus strains and plant extracts for Kazakh population.

Validation of Lactobacillus by Culture and Biochemical tests
Culture and Biochemical properties of all the strains Lactobacillus were done by Gram staining, Catalase and Oxidase test [25][26][27][28] .

Antagonistic activity
Antagonistic activity of Lactobacillus strains was detected by agar well diffusion method on Muller Hinton agar, previously inoculated with 0.1 ml of a 24 h broth culture of indicator microorganisms.The indicator bacteria E. coli ATCC 25922, S. aureus 209P, S. marcescens 221F were incubated in Nutrient Broth (NB, HiMedia, Mumbai, India) at 37°C, and C. albicans ATCC 885-653 in Sabouraud Dextrose Broth (SDB, HiMedia) at appropriate temperature for 24 h.The 100 µl of Lactobacilli inoculum were loaded into each well (diameter 5 mm).Plates were incubated at 37°C for 48 hours.After incubation, all plates were examined for the presence of zone of inhibition around the Wells 29 .

Bacteriocin-producing activity
Study of bacteriocin production by Lactobacillus used the agar diffusion method and the same indicator strains as above.To 15 ml of 0.7% semiliquid Man, Rogosa, Sharp (MRS, HiMedia) agar cooled to 50°C was added 1 ml of indicator culture (10 8 CFU/ml) (McFarland standard set, HiMedia).After solidification, three holes per plate of 5-mm diameter were excavated for each tested Lactobacillus strains and 35 µl of supernatant were added to each well.Supernatants of Lactobacillus strains were prepared as follows: 1 ml of lyophilized culture was added to 20 ml of liquid medium MRS and incubated for 16 h at 37°C, after which 1 ml of the broth from the cell suspension was subcultured into 20 ml of liquid MRS medium and incubated for a further 16 h.Thereafter, the cells were removed by centrifugation at 3,095×g for 5 min (Eppendorf Centrifuge 5810 R, Germany).Supernatant was added to the first well.To eliminate inhibitory activity due to organic acids, the pH of the supernatant was adjusted to pH 6.0 with 1 M NaOH and that solution was added to the second well in a volume of 35 µl.Supernatant with pH 6.0 was added to the third well, along with 1 mg/ml of catalase (Sigma, USA) to remove hydrogen peroxide.The plates were incubated for 1 day.A positive result for the presence of bacteriocin in the supernatant was the presence of an inhibition zone of the indicator strain around the third well 30 .

Antibiotics susceptibility
The susceptibility of the Lactobacillus strains were tested against different antibiotics classes including Penicillin, Cephalosporine, Tetracycline, Glycopeptides, Quinolone, Lincozamids, Carbapenems, Aminoglycoside, Macrolides and others preparations.The bacterial suspension (10 8 CFU/ml) was inoculated onto MRS agar (HiMedia, India) plates using swabbing technique.Then antibiotics disks were deposited on the plates.The susceptibility / resistance to these antibiotics was examined after incubation at 37°C for 24 h the inhibition zones around the disks 31 .

Testing of NaCl tolerance
For the determination of NaCl tolerance of the Lactobacillus strains three test tube containing MRS broth (HiMedia, India) were adjusted with different concentration 2%, 4%, 6%, 10% of NaCl.After sterilization, each test tube inoculated with 1% (v/v) frech over night culture of Lactobacillus strains and incubated at 37°C for 24 h.After 24 h of incubation their growth were determined by observing their turbidity.Maximum growth were indicated as double positive sign (++), normal growth as single positive sign (+) and no growth were indicated as negative sign (-) 32 .

Testing of bile tolerance
The Lactobacillus strains were inoculated in MRS broth (HiMedia, India) supplemented with 0.5%, 1%, 3%, 5%, 10%, 20% of bile bovine (Samson-Med, Russia) along with a control for each of the strains.The cultures were observed for growth for a period of 1 day 33 .

Statistical analysis
Our experiments were performed in triplicate and results elaborated as mean ± standard error of the mean of three experiments.The statistical significance was assessed by Student¼s t test.Results were considered significant at p<0.05.

Antagonistic activity
In recent decades, the selection of microbial molecules and/or bacterial strains able to produce antagonistic molecules to be used as mm.The effect of the three strains of Lactobacillus could be understood from the Figure 2.

Bacteriocin-producing activity
One important attribute of lactic acid bacteria is their ability to produce antimicrobial compounds such as organic acids, diacetyl, hydrogen peroxide, ethanol, reuterin and bacteriocins or bactericidal proteins.In recent years, interest in bacteriocins has grown substantially due to their potential usefulness as natural food preservatives in addition to promoting good health 35 .We investigated bacteriocin-producing activity of culture supernatants of Lactobacillus strains.Figure 3 illustrates typical results of study of bacteriocin production of L. sakei 24a toward E. coli.
It is noteworthy that all the isolates exerted extracellular antimicrobial activity against E. coli ATCC 25922 B-RKM 0447, S. aureus 209 B-RKM 0057, S. marcescens 221F B-RKM 0059 and C. albicans ATCC 885-653 Y B-RKM 0475.Supernatants of Lactobacillus strains no showed antagonism only without addition of alkali, and in front of bacteriocin-producing activity manifested by the addition of catalase, which indicates the presence of bacteriocins in these Lactobacillus strains (Table 1).

Antibiotics susceptibility
Medical treatment with antibiotics can lead to the elimination of essential intestinal microflora and make it an easy target for pathogens.In order to prevent and restore the equilibrium in the gastrointestinal tract it is important to study the susceptibility of the lactobacilli strains to the action of a variety of antibiotics, used in the clinical practice 36 .The susceptibility of Lactobacillus strains were tested against 24 of the most frequently used in medical practice antibiotics with different mechanisms of action.The results of the studies antibiotics susceptibility are summarized in Table 2. L. casei 3 B-RKM 0008 was resistant to all antibiotics from the group Quinolone (ciprofloxacin, ofloxacin, levofloxacin).L. sakei 240 B-RKM 0559 was resistant to all antibiotics from the group Cephalosporine (cefotaxime, ceftriaxone, ceftazidime) with the exception of cefazolin, cefuroxime, cefamandol.From the group of Aminoglycoside the strain L. plantarum 8RA-3 pl B-RKM 0015 was resistant to gentamicin and amicacin.The both strains L. sakei 240 B-RKM 0559 and L. casei 3 B-RKM 0008 were resistant to vancomycin.

Bile and salt tolerance
The ability of Lactobacillus strains to tolerate the effect of different concentrations of bile (0.5-20%) and NaCl (2-10%) after incubation for 24 h tested.The results were shown in Table 3.
From the data in Table 3, all the Lactobacillus strains (L.sakei240, L. plantarum 8RA-3 pl, L. casei 3) were able to survive over the range of 0.5-20% w/v supplementation of bile in MRS broth.The growth of the strains declined with increased supplementation of 20% bile.However, the strain L. sakei 240 B-RKM 0559 showed good growth at 20% bile concentration.
As for NaCl tolerance, all the Lactobacillus strains were able to tolerate 2-6% NaCl except L. sakei 240 B-RKM 0559 which unable to grow at 6%.As for 10% NaCl, all the Lactobacillus strains were unable to grow.

DISCUSSION
The intestine is an extremely complex living system that participates in the protection of the host through a strong defense against aggressions from the external environment.This defensive task is based on 3 constituents that are in permanent contact and dialog with each other: the microflora, mucosal barrier, and local immune system 37 .The use of probiotics is increasing in popularity for the prevention and treatment of intestinal infection and disease.Animal models and human clinical trials indicate that probiotics may reduce intestinal inflammation and alleviate symptoms of colorectal cancer 38 .One of the main components of probiotics are bacteria of the genus Lactobacillus [39][40] .Numerous publications have demonstrated the safety and efficacy of probiotics based on Lactobacillus and Bifidobacteria [41][42] .Moreover, probiotic strains selected by probiotic and technological properties: tolerance to low pH, bile salt, antimicrobial potential, auto-aggregation ability, microbial adhesion to solvents, tolerance to high temperature and osmotic pressure [43][44][45][46][47] .
It is known that the most effective strains that are typical for the region, which can be adapted to the specific macroorganism.Thus, creation and prescription of probiotic preparations should be dependent on the regional group of the population with a special phenotype, genotype, lifestyle, choice of food, environmental conditions [48][49] .
The aim of this work was evaluation the probiotic potential of Lactobacillus strains (L.sakei 240 B-RKM 0559, L. casei 3 -RKM 0008, and L. plantarum 8RA 3-pl B-RKM 0015) for creation combined product for Kazakh population.
The present study showed that L. casei 3 -RKM 0008, L. sakei 24a and L. plantarum 8RA 3pl B-RKM 0015 inhibited growth of pathogenic bacteria, such E. coli, S. aureus, Ser.marcescens.Therefore they are able to prevent the growth of other pathogenic microorganisms in gut system.Furthermore, Georgieva et al. (2015) and also reported antagonistic effect against S. aureus, E. coli, B. cereus and C. albicans 50 .L. casei 3 B-RKM 0008 and L. plantarum 8RA 3-pl B-RKM 0015 were able to inhibit of pathogenic microflora of the urogenital tract with protection against C. albicans as demonstrated by previous studies 51- 52 .
The Bacteriocin activity of the Lactobacillus strains, especially L. sakei 240 B-RKM 0559 against S. aureus shoded a maximum inhibition zone of 9.0±0.58mm at @" 6,0 + catalase.For comparison, Mbawala et al. (2013) in his work to get a maximum inhibition zone of Lactobacillus spp.4.5±0.1 mm 57 .Thereby it was established that Lactobacillus strains had antagonistic activity against pathogenic bacteria and yeast.
Similar results resistance to ciprofloxacin and gentamycin were also observed by Pithva et  al. (2014), resistance to ofloxacin -by Hyacinta et  al. (2015), resistance to amicacin and levofloxacin -by Sharma et al. (2016) where they examined antibiotic resistance of Lactobacillus strains [59][60][61] .But, Haghshenas et al. (2016) shoded that the Lactobacillus plantarum 15HN was sensitive to vancomycin and gentamycin 62 .Antibiotics resistance of Lactobacillus could also be regarded as a beneficial property.A resistant probiotic strain that is co-administered with an antibiotic may reduce the gastrointestinal side effects related to antibiotic treatment 63 .
Bile tolerance is one of the essential properties required for lactic acid bacteria to survive in the small intestine and to be functionally effective intestine 64 .For a probiotic strain to be able to perform effectively in the gastrointestinal tract, it must overcome the antimicrobial challenge posed by bile.In this study, three Lactobacillus strains were tested for bile tolerance.These observations do not agree with those reported by Park et al. (2006) who showed that four strains Lactobacillus acidophilus were slightly suppressed over time and showed bile resistance at 3-5% ox gall 65 .Ours results indicate that Lactobacillus strains selected for probiotic tolerant to bile at a concentration of up to 20%.Tolerance to bovine bile for Lactobacillus spp.has been studied by Elcioglu et al. (2014) and Khagwal et al. (2014) [66][67] .
While sodium chloride is growth inhibitory to several other types of bacteria, the probiotic organisms withstand high salt concentration in the human gut 68 .All the Lactobacillus strains had good growth up to 2-6% concentration in the culture medium, with the exception of L. sakei 240 B-RKM 0559.Our results have the similarities with the findings of Pundir et  al. (2013) that were tolerable to 1-6% NaCl 69 .Bhardwaj et al. (2016) demonstrated that no growth even for a single Lactobacillus strain was noticed at the sodium chloride concentration of 6.5% 70 .Wang et al. (2016) estabilished that increase in the salt concentrations above 6% resulted in decrease of bacterial density L. plantarum ATCC 14917 71 .Tolerance to NaCl is important because the Kazakh national food such kurt, kazy, shuzhuk contain a lot of salt.From the obtained results we can conclude that Lactobacillus strains (L.sakei 240 -RKM 0559, L. casei 3 -RKM 0008, and L. plantarum 8RA 3-pl -RKM 0015) showed potential as a probiotic owing to its antibiotic resistance, antimicrobial potential and tolerance to bile and salt.

CONCLUSION
In this study, we studied of probiotic potential of Lactobacillus strains (L.sakei 24a B-RKM 0559, L. casei 3 B-RKM 0008, and L. plantarum 8RA 3-pl B-RKM 0015).These strains are therefore good candidates for development of biological products for the Kazakh population in view of geographical region-specificity.

Table 1 .
Bacteriocin-producing activity of Lactobacillus strains

Table 2 .
Antibiotic susceptibility of the Lactobacillus strains R -Resistant, S -Sensitive

Table 3 .
Bile and salt tolerance of the Lactobacillus strains