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Eur Food Res TechnolDOI 10.1007/s00217-014-2224-x

ORIgInal PaPER

In vitro evaluation of the suitability potential probiotic of lactobacilli isolates from the gastrointestinal tract of chicken

Zehra Nur Yuksekdag · Nur Sahin · Belma Aslim

Received: 9 October 2013 / Revised: 25 March 2014 / accepted: 27 March 2014 © Springer-Verlag Berlin Heidelberg 2014

Introduction

The recent 50 years, antibiotics have been extensively used in livestock production at therapeutic levels to treat bacte-rial infections, as well as at sub-therapeutic levels to pro-mote growth in poultry industry [1–3]. Many of the anti-biotics used as growth promoters in the livestock industry especially poultry are also utilized in human medicine [3]. after the initiation of pervasive utilize of antibiotics in the livestock industry, principally the poultry industry, the practice can lead to the development of bacterial resistance to the commonplace microbicidal affects of the antibiot-ics [2, 4, 5]. Ever since, the random utilize of antibiotics in poultry industry has become a cause for concern [4]. In view of their potential to diminish enteric disease in poul-try, probiotics are appraised to be a good alternative to uti-lize of antibiotics [2, 6, 7].

Probiotics are live microorganisms that debate a health benefit to in humans and animals [8, 9]. Perhaps the most generally utilized probiotic species belong to the genus Lactobacillus [10]. The abundance in the upper gastroin-testinal tract of humans and animals is lactobacilli, gram-positive rods that belong to the phylum Firmicutes [11, 12]. It is highly differed and comprised too many species [13, 14]. lactobacilli are considerably utilized in food fermen-tation and are well known for their preservative capability as well as for their positive contribution to texture and fla-vor formation in many food products. In addition, several well-characterized probiotic strains relating this genus are utilized by the food and pharmaceutical industries, and new probiotic lactobacilli strains are devised [11, 15].

The use of lactobacilli has been suggested as an effec-tive strategy to decrease infection in chicken. For this rea-son, the aim of this research was to examine some probiotic properties of the strains: some products of fermentation

Abstract Probiotic lactobacilli could be used to decrease the colonization of pathogenic bacteria in chicken and therefore decrease the risk of foodborne illness to consum-ers. The present study was conducted to select appropriate microbial strains for the development of potential probiotic. In experiment 1, 18 strains of lactobacilli isolated from the gastrointestinal tract of chicken were evaluated. The strains were demonstrated for their lactic acid, hydrogen peroxide, and exopolysaccharide productions. For experiment 2, the strains were tested for their acid, bile, antimicrobial activ-ity, and antibiotic resistance levels. among them, Lactoba-cillus delbrueckii ssp. delbrueckii BaZ32, Lactobacillus acidophilus BaZ29, BaZ36, BaZ43, and BaZ63 which produced high EPS were selected to aggregation ability. It is concluded that L. delbrueckii ssp. delbrueckii BaZ32, L. acidophilus BaZ29 confer high tolerance to acid, bile, anti-biotic resistance, high antimicrobial activity, aggregation ability, and EPS production. These strains may be func-tional feed additives as potential probiotic in chicken.

Keywords lactobacilli · Chicken · Exopolysaccharide · Tolerance to low pH and bile · aggregation ability

Z. n. Yuksekdag (*) · n. Sahin · B. aslim Biotechnology laboratory, Department of Biology, Faculty of Science, gazi University, 06500 ankara, Turkeye-mail: zehranur@gazi.edu.tr

n. Sahin e-mail: nursahin06@gmail.com

B. aslim e-mail: baslim@gazi.edu.tr

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(lactic acid, hydrogen peroxide), acid and bile tolerance, antibiotic resistance, antimicrobial activity against patho-gens, exopolysaccharide production, and capacity of aggre-gation to be utilized as potentially probiotic bacteria suit-able for chicken. It may be suggested the use of probiotics as an alternative to the use of antibiotics in chickens.

Materials and methods

Chemical analysis of fermentation by-products

This research comprised 18 strains of lactobacilli, of which these strains were conducted at gazi University, Biotech-nology laboratory Collection for Type Cultures. The 18 lactobacilli species utilized in the research were isolated from gastrointestinal tract of chickens and also identified by molecular method (16S rDna) (data not shown).

To determine the lactic acid production, the strains of lactobacilli incubated in MRS broth for 24 h at 37 °C were determined by using titratable acidity, expressed as per-centage [16].

The hydrogen peroxide (H2O2) production was analyzed spectrophotometrically for lactobacilli strains as defined by gilliland [17]. Measurements were acquired after 24-h incubation period in MRS broth, and the production was monitored at OD400 (Digilab Hitachi U-1800). H2O2 was gauged by using a H2O2 (30 %) obtained from Merck stand-ard curve, performed with concentrations, ranging from 1 to 10 µg/ml [18].

The antimicrobial affects of the lactobacilli strains against Escherichia coli aTCC 11229, E. coli O157:H7, Staphylo-coccus aureus aTCC 25923, Listeria monocytogenes aTCC 7644, Salmonella enteritidis aTCC 13076, Shigella sonnei Mu:57, Pseudomonas aeruginosa aTCC 27853, and Campy-lobacter jejuni aTCC 33291 were assessed through the ‘agar diffusion technique’ [19]. Briefly, the cell-free filtrates was acquired by centrifugation (10,000 rpm for 15 min, and 4 °C) of overnight (16–18 h) culture of strain grown in MRS broth at 37 °C, and the clear supernatant was sterilized by filtra-tion (0.45 µm, Millipore, France). Petri dishes with 20 ml of Mueller–Hinton agar (Merck) for C. jejuni and nutrient agar (Merck) for other pathogen bacteria were arranged, formerly inoculated with 100 µl of a 24-h Campylobacter enrichment broth (Merck) culture of C. jejuni and nutrient broth culture of other pathogen bacteria. The plates were inspected for demonstration of inhibition after incubation at 37 °C for 24 h by measuring the inhibition zone.

EPS production

The production of EPS by lactobacilli cultures was appraised in 24-h culture in MRS broth at 37 °C, according

to the technique designated by Torino et al. [20], using glu-cose as standard [21].

Detection of antibiotic susceptibility

antibiotic susceptibility testing was done on overnight bacterial culture (0.5 McFarland) disposing agar disk dif-fusion method as described by Taheri et al. [22]. Suscep-tibility testing was performed on cystein-MRS agar. The antimicrobials for disk diffusion testing were acquired from Oxoid in the following concentrations: penicillin-g (10 IU), ampicillin (10 μg), streptomycin (10 μg), gen-tamycin (10 μg), chloramphenicol (30 μg), vancomycin (30 μg), rifampicin (5 μg), and kanamycin (30 μg). The resistance and susceptibility were deciphered according to the Clinical and laboratory Standards Institute [23].

low pH and bile salts tolerance tests

The overnight lactobacilli culture in MRS broth was assessed for growing at low pH (1, 2, 3) and bile salts (0.06, 0.15, 0.30 %), as described by Chung et al. [24]. The cell growth was measured spectrophotometrically (Digilab Hitachi U-1800) at 600 nm. Results were conferred in opti-cal density (OD).

aggregation test

For this test, Lactobacillus delbrueckii ssp. delbrueckii BaZ32, Lactobacillus acidophilus BaZ29, BaZ36, BaZ43, and BaZ63 which produced high EPS were selected. aggregation abilities were performed by the method of Vandervoorde et al. [25] using the aggrega-tion percentage. In autoaggregation capabilities, overnight lactobacilli were re-suspended in the phosphate-buffered saline (130 mM sodium chloride, 10 mM sodium phos-phate, pH 7.2). after the bacterial suspensions were adjusted to McFarland 1 and incubated at room temperature and monitored at 4 h. The percentage of autoaggregation was expressed as previous research [9]. In the coaggrega-tion test, equal volumes of cells (500 µl) of various lac-tobacilli strains and E. coli aTCC 11229 and S. enteritidis aTCC 13076 were mixed, incubated at room temperature without agitation, and monitored at 4 h. Coaggregation was calculated as previous research [9].

Statistics

Each test was carried out in duplicate, and the tests were repeated three times. Results are expressed as the mean and standard error mean. SPSS software version 15.0 (SPSS, Chicago, Il, USa) was used for statistical analyses. Con-centration–cell growth (optical density) relationships were

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designated from the correlation and regression coefficients for the tolerance to pH and bile salts.

Results

Chemical analysis of fermentation by-products

The acid levels produced by lactobacilli strains in MRS are exhibited in Table 1. all lactobacilli tested in the present research could titratable acidities. The level of titratable acidities (%) indicated by lactobacilli ranged from 0.42 % (Lactobacillus salivarius ZYn23) to 0.90 % (L. salivarius

ZYn9). L. acidophilus BaZ22, BaZ29, and BaZ43 (0.89 %) strains indicated a higher level of acidity than the other strains.

There were starkly differences between the amounts of H2O2 production (Table 1). The H2O2 of L. salivarius ZYn9 was highest (3.77 µg/ml), while L. delbrueckii ssp. delbrueckii BaZ32 was lowest (0.18 µg/ml).

Eighteen lactobacilli strains obtained from culture collections were evaluated for inhibitory capacities against pathogen bacteria using an agar diffusion tech-nique (Table 2). The size of the inhibition zone differed slightly between pathogen bacteria; however, the mean size of the pathogen bacteria inhibition zone differed greatly among the lactobacilli strains. L. acidophilus BaZ54 and BaZ61 exhibited powerful inhibitory effects to P. aeruginosa aTCC 27853. Whereas all of the lacto-bacilli inhibited the growth of S. sonnei, one strain (L. acidophilus BaZ36) inhibited the growth of L. mono-cytogenes and two strains (L. acidophilus BaZ29 and L. delbrueckii ssp. delbrueckii BaZ32) inhibited the growth of C. jejuni.

EPS production

lactobacilli strains were tested for their EPS production in this research. The high and low production strains of lac-tobacilli are exhibited in Table 1. L. acidophilus ZYn13 strain produced the minimum amount (10.60 mg/l) and L. delbrueckii ssp. delbrueckii BaZ32 strain produced the maximum amount (180 mg/l) of exopolysaccharide. L. acidophilus BaZ29 (168.90 mg/l), BaZ63 (158.42 mg/l), and BaZ54 (155.00 mg/l) strains induced a higher level of EPS production than all the other strains.

Detection of antibiotic susceptibility

antimicrobial disk diffusion susceptibility of the 18 lacto-bacilli strains of chicken origin is summarized in Table 3. according to our results, more strains were resistant to kanamycin (100 %), gentamicin (100 %), rifampicin (95 %), streptomycin (72 %), and vancomycin (61 %). a

Table 1 Metabolic activities of lactobacilli strains of chicken origin

Strains lactic acid (%) H2O2 (μg/ml) EPS production (mg/l)

L. acidophilus

BaZ22 0.89 ± 0.00 1.36 ± 0.02 72.10 ± 0.02

BaZ29 0.89 ± 0.00 1.56 ± 0.03 168.90 ± 0.01

BaZ36 0.81 ± 0.00 1.90 ± 0.00 135.98 ± 0.01

BaZ43 0.89 ± 0.00 2.10 ± 0.02 140.85 ± 0.02

BaZ51 0.82 ± 0.00 2.12 ± 0.01 124.27 ± 0.00

BaZ54 0.87 ± 0.00 0.92 ± 0.01 155.00 ± 0.01

BaZ59 0.85 ± 0.01 0.49 ± 0.00 107.19 ± 0.01

BaZ61 0.85 ± 0.01 1.72 ± 0.01 102.80 ± 0.03

BaZ63 0.81 ± 0.00 0.89 ± 0.01 158.42 ± 0.02

ZYn13 0.87 ± 0.01 2.31 ± 0.01 10.60 ± 0.01

L. delbrueckii ssp. delbrueckii

BaZ32 0.82 ± 0.00 0.18 ± 0.01 180.36 ± 0.01

ZYn31 0.54 ± 0.01 0.49 ± 0.00 36.47 ± 0.02

ZYn33 0.68 ± 0.01 3.51 ± 0.01 125.85 ± 0.01

L. salivarius

ZYn9 0.90 ± 0.00 3.77 ± 0.02 151.10 ± 0.05

ZYn15 0.85 ± 0.01 2.24 ± 0.02 125.00 ± 0.04

ZYn23 0.42 ± 0.01 1.21 ± 0.04 53.05 ± 0.00

L. rhamnosus BaZ78

0.78 ± 0.01 1.07 ± 0.04 99.63 ± 0.00

L. fermentum ZYn17

0.43 ± 0.00 0.44 ± 0.03 151.70 ± 0.01

Table 2 Inhibitory activity of lactobacilli strains of chicken origin against pathogens responsible of infections in chicken (%)

–, no inhibition detected

E. coliO157:H7

E. coliaTCC 11229

P. aeruginosaaTCC 27853

S. aureusaTCC 25923

L. monocytogenesaTCC 7644

S. enteritidisaTCC 13076

S. sonneiMu:57

C. jejuniaTCC 33291

L. acidophilus (10) 100 100 100 90 10 100 100 10

L. delbrueckii ssp. del-brueckii (3)

33.3 33.3 66.7 33.3 – 33.3 100 33.3

L. salivarius (3) 66.7 66.7 100 66.7 – 66.7 100 –

L. rhamnosus (1) 100 100 100 – – – 100 –

L. fermentum (1) 100 – 100 – – – 100 –

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low prevalence of chloramphenicol resistance was detected in lactobacilli strains (17 %). all strains were intermediate resistance to ampicillin.

low pH and bile salts tolerance tests

Table 4 portrays cell growth (optical density) of lactoba-cilli strains at low pH (1, 2, 3) and bile salts (0.06, 0.15, 0.30 %). all of the lactobacilli strains were able to grow at both low pH and different bile concentrations.

aggregation test

Five lactobacilli strains sharing high EPS production were selected and determined percentage of autoaggregation and coaggregation with E. coli aTTC 11229 and S. ente-ritidis aTCC 13076. among the selected strains, only L. acidophilus BaZ36 exhibited a high autoaggregative pat-tern (33.3 %) (Fig. 1). In addition, the coaggregation per-centage of L. acidophilus BaZ36 with the E. coli aTTC 11229 exhibited the highest percentage (28.3 %), while the coaggregation percentage of L. delbrueckii ssp. delbrueckii BaZ32 with S. enteritidis aTCC 13076 indicated the high-est percentage (25.0 %).

Discussion

The role of probiotics in the treatment of infections is pro-gressively being credentialed as an alternative or comple-ment to antibiotics, with the potential to diminish the use of antibiotics in the poultry industry or decrease their side effects. Probiotics can preclude or diminish antibiotic-asso-ciated side effects and have an inhibitory effect on patho-gen bacteria [2, 3]. Probiotics are added to animal diet to encourage a well-balanced gastrointestinal tract microbi-ota, eventually contributing to enhanced health [26]. They are viable single or mixed cultures of microorganisms that when given to animals or humans, beneficially affect the host by improving the properties of the indigenous micro-biota [27, 28].

lactic acid bacteria (laB), especially Lactobacillus genus, are the most crucial unique probiotic microorgan-isms ordinarily relevant to the healthy intestinal microbi-ota of chicken. The laB population levels in the chicken digestive tract are higher than 108–109 viable cells per gram [29–31]. The dominance of laB species can vary depend-ing on the anatomical site [30, 32].

lactic acid is of prime crucial in feed technology, where their essential role is inhibition of growth of pathogen

Table 3 antibiotic susceptibility of lactobacilli strains of chicken origin

R resistant, I intermediate resistance, S susceptible

Strains Penicillin-g ampicilin Streptomycin gentamicin Chloramphenicol Vancomycin Rifampicin Kanamycin

L. acidophilus

BaZ22 I I R R I I R R

BaZ29 I I R R I R R R

BaZ36 S I I R I I R R

BaZ43 I I R R I R R R

BaZ51 I I R R I R R R

BaZ54 I I I R I R R R

BaZ59 I I R R I R R R

BaZ61 S I I R I R R R

BaZ63 I I R R I R R R

ZYn13 I I I R I R R R

L. delbrueckii ssp. delbrueckii

BaZ32 I I I R I R R R

ZYn31 I I R R R I R R

ZYn33 I I R R I I I R

L. salivarius

ZYn9 I I R R I R R R

ZYn15 I I R R I I R R

ZYn23 S I R R R I R R

L. rhamnosus

BaZ78 S I R R R I R R

L. fermentum

ZYn17 I I R R I R R R

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microorganisms. The production of high levels of lactic acid has positive effect on human and animal health. lac-tobacilli are capable of producing and excreting inhibitory substances other than lactic acid. These matters included formic acid, free fatty acids, ammonia, hydrogen peroxide,

diacetyl, bacteriolytic enzymes, bacteriocins, and antibiot-ics as well as several well designated or thoroughly undes-ignated inhibitory substances are antimicrobial activity to a wide spectrum of microorganism [31, 33]. according to the results obtained here, L. salivarius ZYn9 produced the highest amounts of both lactic acid and hydrogen peroxide (Table 1).

Probiotic lactobacilli can be used to diminish the colo-nization of pathogenic bacteria in food animals and thus diminish the risk of foodborne illness to consumers. Prob-able mechanisms for the reduction in pathogens by lacto-bacilli enclose: (1) stimulation of adaptive immunity; (2) alteration of the cecal microbiome; and (3) production of antagonistic metabolites, such as organic acids [10]. The research’s reported that lactobacilli have been exhibited to inhibit the growth of various bacterial pathogens by the production of organic acids, hydrogen peroxide, and spe-cific inhibitory proteins called bacteriocins and/or bacteri-ocin-like substances [9, 34].

antimicrobial activity by probiotic lactobacilli cul-tures has been denoted in vitro [35, 36]. Therefore, Zhang

Table 4 Tolerance to low pH and bile salts of lactobacilli strains of chicken origin and regression coefficients

a Highest and lowest optically densityb Control: pH 6.0 and without bile salts

pHa Bile concentrationsa

Strains Controlb 1.0 2.0 3.0 Multi-ple R

Controlb 0.06 % 0.15 % 0.30 % Multi-ple R

L. acidophilus

BaZ22 2.523 ± 0.00 0.143 ± 0.00 0.169 ± 0.00 0.410 ± 0.01 0.957 2.569 ± 0.02 2.530 ± 0.00 2.383 ± 0.01 1.322 ± 0.00 −0.936

BaZ29 2.620 ± 0.02 0.146 ± 0.00 0.168 ± 0.00 0.339 ± 0.00 0.948 2.649 ± 0.01 2.610 ± 0.01 2.527 ± 0.00 2.055 ± 0.03 −0.953

BaZ36 2.568 ± 0.00 0.130 ± 0.00 0.155 ± 0.00 0.389 ± 0.00 0.955 2.593 ± 0.03 2.550 ± 0.00 2.443 ± 0.02 1.779 ± 0.01 −0.947

BaZ43 2.456 ± 0.05 0.143 ± 0.03 0.175 ± 0.00 0.365 ± 0.01 0.953 2.537 ± 0.02 2.529 ± 0.01 2.341 ± 0.04 1.153 ± 0.00 −0.933

BaZ51 2.468 ± 0.00 0.144 ± 0.02 0.172 ± 0.02 0.350 ± 0.00 0.951 2.502 ± 0.01 2.495 ± 0.01 2.423 ± 0.00 1.471 ± 0.00 −0.911

BaZ54 2.620 ± 0.00 0.151 ± 0.02 0.168 ± 0.01 0.378 ± 0.00 0.952 2.553 ± 0.00 2.325 ± 0.00 2.076 ± 0.01 1.463 ± 0.02 −0.997

BaZ59 2.678 ± 0.04 0.148 ± 0.01 0.166 ± 0.03 0.383 ± 0.01 0.952 2.531 ± 0.02 2.377 ± 0.04 2.319 ± 0.00 1.293 ± 0.03 −0.940

BaZ61 2.638 ± 0.00 0.145 ± 0.00 0.158 ± 0.00 0.362 ± 0.02 0.950 2.592 ± 0.01 2.488 ± 0.03 2.366 ± 0.01 1.789 ± 0.00 −0.973

BaZ63 2.602 ± 0.03 0.165 ± 0.00 0.153 ± 0.00 0.407 ± 0.00 0.956 2.678 ± 0.03 2.602 ± 0.01 2.553 ± 0.00 2.357 ± 0.01 −0.989

ZYn13 2.678 ± 0.00 0.140 ± 0.01 0.153 ± 0.00 0.390 ± 0.02 0.953 2.530 ± 0.00 2.505 ± 0.02 2.362 ± 0.00 1.795 ± 0.01 −0.959

L. delbrueckii ssp. delbrueckii

BaZ32 2.568 ± 0.01 0.141 ± 0.01 0.164 ± 0.01 0.349 ± 0.02 0.950 2.553 ± 0.00 2.523 ± 0.00 2.498 ± 0.03 2.293 ± 0.00 −0.954

ZYn31 2.620 ± 0.04 0.127 ± 0.01 0.175 ± 0.03 0.380 ± 0.01 0.954 2.577 ± 0.04 2.557 ± 0.01 2.507 ± 0.03 2.123 ± 0.02 −0.937

ZYn33 2.731 ± 0.00 0.133 ± 0.01 0.158 ± 0.00 0.365 ± 0.02 0.951 2.620 ± 0.03 2.585 ± 0.00 2.523 ± 0.00 1.917 ± 0.01 −0.932

L. salivarius

ZYn9 2.745 ± 0.00 0.144 ± 0.01 0.154 ± 0.01 0.365 ± 0.04 0.950 2.517 ± 0.01 2.450 ± 0.01 2.367 ± 0.02 2.028 ± 0.00 −0.979

ZYn15 2.658 ± 0.00 0.142 ± 0.00 0.156 ± 0.00 0.380 ± 0.00 0.950 2.602 ± 0.02 2.553 ± 0.04 2.495 ± 0.01 1.598 ± 0.00 −0.921

ZYn23 1.735 ± 0.05 0.135 ± 0.00 0.152 ± 0.01 0.389 ± 0.00 0.966 1.735 ± 0.00 1.725 ± 0.00 1.712 ± 0.00 1.494 ± 0.03 −0.917

L. rhamnosus

BaZ78 2.620 ± 0.02 0.135 ± 0.01 0.152 ± 0.02 0.400 ± 0.03 0.955 2.516 ± 0.01 2.495 ± 0.00 2.425 ± 0.00 1.958 ± 0.03 −0.940

L. fermentum

ZYn17 1.477 ± 0.10 0.119 ± 0.02 0.149 ± 0.00 0.375 ± 0.01 0.973 1.684 ± 0.02 1.620 ± 0.03 1.607 ± 0.02 1.522 ± 0.00 −0.972

0

5

10

15

20

25

30

35

BAZ29 BAZ36 BAZ43 BAZ63 BAZ32

Autoaggregation %Coaggregation % with E. coliCoaggregation % with Salmonella enteritidis

Fig. 1 autoaggregation and coaggregation scores (%) for lactobacilli strains of chicken origin

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et al. [37] reported that a number of L. salivarius strains exhibited in vitro activity against Salmonella and C. jejuni. Based on our results, while all lactobacilli strains inhibited the growth of S. sonnei Mu:57 (100 %), only L. delbrueckii ssp. delbrueckii BaZ32, and L. acidophilus BaZ29 inhib-ited the growth of C. jejuni. The 10–100 % of lactobacilli strains shown inhibitory activity against pathogen bacteria (Table 2).

among the wide variety of polysaccharide producing microorganisms, laB have attained special care because of the notable property of the polymers they synthesize and as they do not relocate any health risk which are generally regarded as safe (gRaS). In gastrointestinal tract, EPS from laB will stay stable in order to increase the coloniza-tion of probiotic bacteria. EPS produced by laB is either as capsular polysaccharides (CPSs) or slime polysaccha-rides. The CPSs powerfully bind with bacterial cell surface, while slime EPS is secreted into surrounding environment [31, 38]. aslim et al. [39] reported that the EPS produced by lactobacilli strains during growth in MRS medium ranged between 21 and 211 mg/l. Our results indicated that lactobacilli strains produce comparatively higher quantities of EPS and were similar to findings of other researchers. although all the lactobacilli produced exopol-ysaccharide in high amounts, only L. acidophilus ZYn13 (10.60 mg/l) and L. delbrueckii ssp. delbrueckii ZYn31 (36.47 mg/l) were low producers (Table 1). Furthermore, both the type and concentration of nitrogen sources in the growth medium, environmental conditions as temperature, pH, incubation time, and source of isolation are known to impress the quantity of EPS produced by laB [9, 39–43].

Horizontal gene transfer to different bacteria in the gastrointestinal tract could cause the development of new antibiotic resistant microorganisms [44, 45]. a crucial requirement for probiotic strains is that they should not carry transmissible antibiotic resistance genes [44, 46]. In the present research, 18 lactobacilli strains were tested for antibiotic susceptibility against 8 antimicrobial com-pounds. agar disk diffusion method enabled to reveal mod-erately susceptible for penicillin-g (78 %). The most effec-tive antimicrobials were gentamicin, kanamycin (100 % of strains inhibited), and rifampicin (95 % of strains inhibited) (Table 3).

Beneficial effects of probiotics bacteria are only attain-able if microorganisms are able to survive to inadequate conditions in the gastrointestinal tract, such as low pH, as well as high concentration of bile salts [44, 47]. The pre-sent studied strains exhibited a good adaptation to expo-sure at low pH (1, 2, 3) values and bile salts concentration (0.06, 0.15. 0.30 %), especially at pH 3 and 2, L. acido-philus BaZ22 strain indicated growth ratio of 0.410 OD after treatment with pH 3, while L. acidophilus BaZ43 strain shown growth ratio of 0.175 OD, after treatment at

pH 2. as concern the resistance at pH 1, the determined growth ratio ranged from 0.119 to 0.151 OD. L. acido-philus BaZ29, BaZ59, BaZ61, and L. delbrueckii ssp. delbrueckii BaZ32 strains showed a higher level of tol-erance than all the other strains (Table 4). Our results are in accordance with Messaoudi et al. [2], Bosch et al. [48], Zago et al. [49] and Yuksekdag and aslim et al. [9]. On the contrary, Yamazaki et al. [34] and Klayraung et al. [50] highlight the capability of lactobacilli strains to not survive at low pH and bile salts. Comparison among data on toler-ance to low pH and bile salts of lactobacilli strains from different authors presents that these characteristics are strain and isolation resource related. The growth (optical density) significantly diminished with reducing concentra-tion of pH values and increasing concentration of bile. The decrease was significant in all pH/bile salt concentrations and growth (optical density). For all the strains, multiple R values are presented in Table 4.

In general, results evaluated that L. acidophilus BaZ29, BaZ36, BaZ43, BaZ63, and L. delbrueckii ssp. del-brueckii BaZ32 were able to grown at low pH, different bile salts, and produced the highest amounts of exopolysac-charide. Therefore, these strains were selected for aggrega-tion ability.

autoaggregation and coaggregation with certain patho-genic bacteria ability are another reliable criterion for the selection of probiotic [2, 51]. Infections by using lactoba-cilli with probiotic properties adherence to epithelial cells and that coaggregation lead to formation of a barrier that prevents colonization by pathogens [52]. In the present research, L. acidophilus BaZ36 and L. delbrueckii ssp. del-brueckii BaZ32 strains indicated a higher level of autoag-gregation ability than the other strains (Fig. 1). The results recommend that these lactobacilli strains have the ability to aggregate in the gastrointestinal tract. Heravi et al. [53] informed 39 lactobacilli isolated from the gastrointestinal tract of broiler chickens have the ability to autoaggregation activity, in agreement with the present results. Heravi et al. [53] also reported that it is reasonable to assume that bacte-ria with autoaggregation ability have higher hydrophobicity and adhesion.

Salmonella and E. coli are widespread pathogenic bac-teria that menace the safety of gastrointestinal tract in poultry [53]; therefore, in coaggregation study, these path-ogens were selected. Coaggregation between the lactoba-cilli strains and E. coli aTTC 11229/S. enteritidis aTCC 13076 shows low percentage (Fig. 1). For coaggregation ability with pathogen bacteria, we observed a trend simi-lar to those reported by Hutari et al. [54]. gusils et al. [55] emphasized that coaggregation properties might be thought to be linked to the ability to interact closely with undesira-ble bacteria. lactobacilli which showed high coaggregation ability might be thought to be very potential as probiotic

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for chicken. In addition, coaggregation ability may declare that lactobacilli have the ability to trap pathogen bacteria in gastrointestinal tract. The results demonstrate that most of lactobacilli strains of chicken origin were able to inhibit the growth of E. coli aTCC 11229 and S. enteritidis aTCC 13076 and survive simulated passage through the gastro-intestinal tract. The selected lactobacilli strains could act in the lower gastrointestinal tract to prevent diseases in chicken.

This present research designated that among all of the screening methods, there were only significant differences in the results of resistance to low pH and bile salts, suscep-tibility to antibiotics, inhibition of pathogens, EPS produc-tion, and aggregation ability. Our results highlight that L. delbrueckii ssp. delbrueckii BaZ32, L. acidophilus BaZ29 may be fulfill the principle requirements of an efficient pro-biotic and may be seen as reliable candidates for further validation studies in chicken.

Acknowledgments This research was funded by gazi Univer-sity Scientific Research Projects Department project coded with 05/2010-49.

Conflict of interest none.

Compliance with Ethics Requirement This article does not con-tain any studies with human or animal subjects.

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