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

Zinigul Sarmurzina1, Gulmira Bissenova2, Kunsulu Zakarya3Raikhan Dospaeva4, Serik Shaikhin5 and Akhan Abzhalelov6

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).

DOI: http://dx.doi.org/10.22207/JPAM.11.1.20

Abstract

The health status of the individuals is determined by the microflora inhabiting the gastrointestinal tract. One way to restore the balance of the gastrointestinal microflora is the use of probiotics on the Lactobacillus strains. Probiotic bacteria must have a set of properties that allow them to compete with pathogens and opportunistic pathogens in the gut. Antimicrobial activity, antibiotic susceptibility, tolerance to bile and salt of Lactobacillus strains (Lactobacillus casei 3 B-RKM 0008, Lactobacillus plantarum 8RA-3 pl B-RKM 0015, Lactobacillus sakei 24a B-RKM 0559) we studied. The results showed that L. casei 3 B-RKM 0008 and L. plantarum 8RA-3 pl B-RKM 0015 showed antagonism toward conditionally pathogenic microorganisms, including Escherichia coli ATCC 25922 B-RKM 447, Staphylococcus aureus 209P B-RKM 0057, Serratia marcescens 221F B-RKM 0059, and Candida albicans ATCC 885-653 Y-RKM 0475. Strains of Lactobacillus were resistant to gentamicin, ofloxacin, ciprofloxacin, levofloxacin, cefotaxime, ceftriaxone, ceftazidime, amicacin. Lactobacillus strains to be able to grow in the bile (0.5-20%) and salt (2-6%). These results suggest that these strains may be used in the future as probiotic cultures for manufacturing of novel synbiotic product.

 Keywords: Lactobacillus, Antimicrobial activity, Antibiotic, Bile, Salt.

Introduction

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-allergicprocessesand 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 tract5. 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.

Within the latter genus several species are currently used as probiotics, including Lactobacillus acidophilus, Lactobacillus casei/paracasei, Lactobacillus fermentum, Lactobacillus johnsonii, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus rhamnosusand Lactobacillus salivarius8.

Probiotic potential included survival in gastrointestinal simulated juice, antagonistic and bacteriocin activity, acid pH and bile tolerance, antibiotic resistance to antibiotics, adhesion ability and others9-11.

The genera Lactobacillus, Lactococcus, Leuconostoc, Pediococcus, and Enterococcus have been associated with more than 300 different bacteriocins. The inhibition spectra of the bacteriocinsproduced 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 Salmonella12-14.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 bacteria16.Moreover, Lactobacilli, PediococciandLeuconostoc spp. have been reported to be highly resistant to vancomycinand some Lactobacilli have high resistance to bacitracin, cefoxitin, ciprofloxacin, fusidic acid, streptomycin, sulphadiazine, teicoplanin,vancomycin, erythromycin, gentamicin and kanamycin 17-20.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 forprobiotic strains 22-23.Different stress factors might considerably affect viability or performance of the Lactobacillus strains24.

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.

Materials and Methods

Bacterial strains

Lactobacillus casei 3 В-RKM 0008, Lactobacillus plantarum 8RA 3-pl В-RKM 0015, Lactobacillus sakei 24а В-RKM 0559and Indicatormicroorganisms, suchEscherichia coli АТСС 25922 В-RКМ 0447, Staphylococcus aureus209Р В-RКМ 0057,Serratiamarcescens221F В-RКМ 0059,and Candida albicansАТСС 885-653 Y-RКМ 0475, were purchased from Republican Collection of Microorganisms, Ministry of Education and Science of the Republic of Kazakhstan (Astana, Kazakhstan).

 Validation of Lactobacillus by Cultureand Biochemical tests

Culture and Biochemical properties of all the strains Lactobacillus were done by Gram staining, Catalase andOxidase test25-28.

 Antagonistic activity

Antagonistic activityof 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 АТСС 25922, S. aureus209Р,S. marcescens221Fwere incubated in Nutrient Broth (NB, HiMedia, Mumbai, India) at 37°C, and C. albicansАТСС 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 Wells29.

 Bacteriocin-producing activity

Study of bacteriocin production by Lactobacillus used the agar diffusion methodand 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 (108 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 well30.

Antibiotics susceptibility

The susceptibility of the Lactobacillus strains were tested against different antibiotics classes includingPenicillin,Cephalosporine, Tetracycline, Glycopeptides, Quinolone,Lincozamids,Carbapenems,Aminoglycoside,Macrolides and others preparations.The bacterial suspension (108 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) frechover 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 day33.

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 Studentʼst test.Results were considered significant at p˂0.05.

Results

Three strains of Lactobacillus sakei24а В-RKM 0559, isolated from Kazakh national productkazy, Lactobacillus casei3 В-RKM 0008, isolated from human fecal,and Lactobacillus plantarum8RA 3-pl В-RKM 0015, isolated fromcommercial preparation «Lactobacterin» were studied. All the strains of Lactobacillus were validated for their morphological and biochemical characteristics: Gram positive rods(Figure 1), No-spore, Catalase and Oxidase negative test.Colonies on medium MRS agar (HiMedia, India) are smooth, round, shiny, color cream, with straight edge, convex profile, texture thick, diameter 1.0-3.0 mm.

Figure 1 – Morphology of Lactobacillus strains under light microscope after Gramʼs reaction, 100×

1 – L.casei 3 B-RKM 0008, 2 –L. plantarum8RA-3 pl B-RKM 0015, 3 – L. sakei 24аВ-RKM 0559

Antagonistic activity

In recent decades, the selection of microbial molecules and/or bacterial strains able to produce antagonistic molecules to be used as antimicrobials and preservatives has been attracting scientific interest, in order to eliminate or reduce chemical additives34.The antagonistic activity of the threeLactobacillusstrains (L. sakei24а, L. plantarum 8RA-3 pl, L. casei 3) was screened against E. coli АТСС 25922 В-RКМ 0447, S. aureus209Р В-RКМ 0057, S. marcescens221F В-RКМ 0059 andC. albicans АТСС 885-653 Y-RКМ 0475were determined by measuring the zone of inhibition.L. casei 3 B-RKM 0008 and L. plantarum 8RA-3 pl were able to inhibit all the testing pathogenic microorganisms such as E. coli, S. aureus, S. marcescens and C. albicans. Whereas,L. sakei24a failed to show effect over C. albicans.The strain L. casei 3 B-RKM 0008 had high antagonistic activity toward all investigated test strains; the diameter of the zones of inhibition was range 10-13 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 ofL. sakei 24a toward E. coli.

Figure 3 – Bacteriocin activityof L. sakei 24aВ-RKM 0559

It is noteworthy that all the isolates exerted extracellular antimicrobial activity against E. coli АТСС 25922 В-RКМ 0447, S. aureus209Р В-RКМ 0057, S. marcescens 221F В-RКМ 0059 and C. albicans АТСС 885-653 Y-RКМ 0475.Supernatants of Lactobacillus strains no showed antagonism only without addition of alkali, and in front of bacteriocin-producing activitymanifested by the addition of catalase, which indicates the presence of bacteriocins in these Lactobacillus strains (Table 1).

Table 1Bacteriocin-producing activity of Lactobacillus strains

Test-cultures Variant Bacteriocin-producing activity of LAB (mm)
L. casei 3B-RKM0008 L. plantarum 8RA-3 plB-RKM0015 L. sakei24а

B-RKM0559

E. coli* control 7.0±1.08 6.0±0.58 8.0±0.71
+ NaOHpH 6,0
рН 6,0 +catalase 6.0±0.13 6.0±1.0 8.0±0.58
S. aureus** control 7.0±0.71 6.0±0.58 8.0±0.91
+ NaOHpH 6,0
рН 6,0 +catalase 6.0±0.58 6.0±1.0 9.0±0.58
Ser. marcescens control 6.0±0.41 6.0±0.91 6.0±0.58
+ NaOHpH 6,0
рН 6,0 +catalase 6.0±0.58 7.0±0.41 6.0±1.0
C. albicans*** control 7.0±0.91 7.0±0.82 6.0±1.0
+ NaOHpH 6,0
рН 6,0 +catalase 7.3±0.33 7.0±0.58 6.0±0.41
*Р˂0,02

**P˂0,5

***P˂0,1

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 practice36.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.

Table 2Antibiotic susceptibility of the Lactobacillus strains

Class Antibiotics Quantity Antibiotic susceptibility
L. sakei24а   В-RKM 0559 L. plantarum8RA 3-pl В-RKM 0015 L. casei3    В-RKM 0008
Penicillin Ampicillin 25 µg/disc S S S
Amoxycillin 25 µg/disc S S S
Piperacillin 30 µg/disc S S S
Carbenicillin 100 µg/disc S S S
Quinolone Ciprofloxacin 30 µg/disc S S R
Ofloxacin 5 µg/disc S S R
Levofloxacin 5 µg/disc S S R
Lincosamid Lincomicin 15 µg/disc S S S
Carbapenem Meropenem 10 µg/disc S S S
Imipenem 10 µg/disc S S S
Glycopeptid Vancomycin 30 µg/disc R S R
Cephalosporine Cefazolin 30 µg/disc S S S
Cefuroxime 30 µg/disc S S S
Cefamandol 30 µg/disc S S S
Cefotaxime 30 µg/disc R S S
Ceftriaxone 30 µg/disc R S S
Ceftazidime 30 µg/disc R R S
Aminoglycoside Amicacin 30 µg/disc S R R
Gentamicin 30 µg/disc S R S
Macrolide: Erythromycin 15 µg/disc S S S
Clarithromycin 30 µg/disc S S S
Azithromycin 30 µg/disc S S S
Tetracycline: Doxycycline 30 µg/disc S S S
Others: Linezolid 30 µg/disc S S S
R – Resistant, S  -Sensitive

 

 

 

  1. casei 3B-RKM0008 was resistant to all antibiotics from the group Quinolone (ciprofloxacin, ofloxacin, levofloxacin). L. sakei24а B-RKM0559 was resistant to all antibiotics from the group Cephalosporine (cefotaxime, ceftriaxone, ceftazidime) with the exception ofcefazolin, cefuroxime, cefamandol. From the group of Aminoglycoside the strain L. plantarum 8RA-3 plB-RKM0015was resistant togentamicin and amicacin. The both strains L. sakei24а B-RKM0559 and L. casei 3B-RKM0008 were resistant to vancomycin.

 Bile and salt tolerance

The ability of Lactobacillus strainsto tolerate the effect of different concentrations of bile (0.5-20%) and NaCl (2-10%) after incubationfor 24 h tested. The results were shown in Table 3.

Table 3Bile and salt toleranceof the Lactobacillus strains

Tolerance Lactobacillus strains
L. casei 3B-RKM0008 L. plantarum 8RA-3 plB-RKM0015 L. sakei24а

B-RKM0559

Bile                          0.5%

1%

3%

5%

10%

20%

++ ++ ++
++ ++ ++
++ ++ ++
++ ++ ++
++ ++ ++
+
Salt 2% ++ ++ ++
4% + + +
6%

10%

+ +
++, good growth

+, visible growth

-, no growth

From the data in Table 3, all the Lactobacillus strains (L. sakei24а, 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. sakei24а B-RKM0559 showed good growth at 20% bile concentration.

As for NaCl tolerance, all the Lactobacillus strains were able to tolerate 2-6% NaCl except L. sakei24а B-RKM0559 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 cancer38.One of the main components of probiotics are bacteria of the genus Lactobacillus39-40. Numerous publications have demonstrated the safety and efficacy of probiotics based on Lactobacillus and Bifidobacteria41-42.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 43-47.

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 conditions48-49.

The aim of this work was evaluation the probiotic potential of Lactobacillus strains (L.sakei24а В-RKM 0559,L.casei3 В-RKM 0008,and L.plantarum8RA 3-pl В-RKM 0015) for creation combined product for Kazakh population.

The present study showed that L.casei3 В-RKM 0008,L. sakei24аand L.plantarum8RA 3-pl В-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. albicans50. L.casei3 В-RKM 0008 and L.plantarum8RA 3-pl В-RKM 0015were able to inhibitof pathogenic microfloraof the urogenital tract with protection against C. albicansas demonstrated by previous studies 51-52.

Bacteriocin production, along with the production of for example, lactic acid, hydrogen peroxide, and lysozyme, relates to antagonism 53-54.L. casei3 В-RKM 0008,L. plantarum8RA 3-pl В-RKM 0015 and L. sakei24a B-RKM 0559 possessed antagonistic activity against Gram-positive bacteria (S. aureus), Gram-negative bacteria (E. coli, Ser. marcescens)andYeast (C. albicans). Earlier studies reported that bacteriocinsnot all of lactic acid bacteria are inefficient to inhibitE.coli because the outer membrane hinders the site for bacteriocin action55-56.

The Bacteriocin activity of the Lactobacillus strains, especiallyL. sakei24а B-RKM0559 againstS. aureusshoded a maximum inhibition zone of 9.0±0.58mm atрН 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 mm57. Thereby it was established that Lactobacillus strains had antagonistic activity against pathogenic bacteria and yeast.

Antimicrobial resistance in microbes poses a global and increasing threat to public health 58. The Lactobacillus strains were found resistant to clinically relevant antibiotics to cure infections. Almost all of the tested strains (L. casei 3B-RKM0008, L. plantarum 8RA-3 plB-RKM0015, L. sakei24а B-RKM0559) were resistant to ciprofloxacin, ofloxacin, levofloxacin, vancomycin, cefotaxime, ceftriaxone, ceftazidime, amicacin and gentamycin.

Similar results resistance to ciprofloxacin and gentamycinwere also observed by Pithvaet al. (2014), resistance to ofloxacin – by Hyacintaet al. (2015), resistance to amicacin and levofloxacin  – by Sharmaet al. (2016)where they examined antibiotic resistance ofLactobacillus strains59-61.But, Haghshenaset al. (2016) shodedthat the Lactobacillusplantarum 15HNwas sensitive tovancomycin and gentamycin62.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 showedthat four strains Lactobacillus acidophilus were slightly suppressed over time and showed bile resistance at 3-5% oxgall65. 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 exceptionof L. sakei24а B-RKM0559.Our results have the similarities with the findings of Pundir et al. (2013)that were tolerable to 1-6% NaCl69.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 1491771. 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. sakei24а В-RKM 0559,L. casei3 В-RKM 0008,and L. plantarum8RA 3-pl В-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. sakei24а В-RKM 0559,L. casei3 В-RKM 0008,and L. plantarum8RA 3-pl В-RKM 0015). These strains are therefore good candidates for development of biological products for the Kazakh population in view of geographical region-specificity.

Acknowledgements

This work was financially supported by the Scientific Committee of the Ministry of Education and Science of Republic of Kazakhstan (Project number GF 0982).

 

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