Molecular Characterization of Bacteriocinogenic , Antifungal and Probiotic Lactic Acid Bacteria Isolated from Chicken Gastrointestinal Tract

In this study, bacteriocinogenic Lactobacillus plantarum isolates capable of inhibiting foodand feed-borne filamentous fungi from the gastrointestinal tract (GIT) of broiler chicken were identified using 16S rRNA gene sequencing and further evaluated for probiotic properties in vitro. Six potent lactobacilli were selected from one hundred and thirteen isolates for the present study based on their ability to inhibit both pathogenic bacteria and filamentous fungi. They were characterized using various physiological, biochemical and molecular methods. They were acetoin producers, homo fermentative, catalase-negative and producing racemic lactic acid (10 20 mM). All the six isolates exhibited varied sugar utilization and RAPD pattern, indicated their strain level genotypic variation. The 16S rRNA gene sequence and multiplex PCR analysis confirmed that these isolates were Lactobacillus plantarum. The isolates being resistant to low pH (2.0) and bile salt (0.6%) could survive in the gastrointestinal tract of host indicating their potential probiotic application. The isolates were non-pathogenic (γ-hemolytic) and exhibited resistance to antibiotics ciprofloxacin, nalidixic acid, norfloxacin, nitrofurantoin, colistin and streptomycin. They demonstrated strong autoaggregating phenotype ranging from 78% to 86% and showed 49% 61% and 30% 46% coaggregation with E. coli MTCC 728 and L. monocytogenes MTCC 657, respectively. The percentage of hydrophobicity ranged from 16% 33% for all the isolates showing that surface was rather hydrophilic. They exhibited β-galactosidase activity ranging from 1036 1179 MU, bile salt hydrolase activity assisting to reduce serum cholesterol and produced the anti-Listerial bacteriCorresponding author. V. Nallala, K. Jeevaratnam 645 ocin. The strong inhibitory activity of these isolates against food spoilage molds and bacteria with probiotic properties indicates their potential application as food preservatives.


Introduction
Molds are frequent and important spoilage organisms in the food and feed systems, causing huge economic losses to industrials and health hazards to consumers [1].The major species involved in the food spoilage are Aspergillus, Fusarium, Penicillium and Mucor.Chemical preservatives such as propionic, sorbic and acetic acids and their salts only decrease fungal infections and fall short of contaminant elimination.Additionally, some molds have acquired the ability to degrade chemical preservatives such as sorbate [2].The raising consumer concerns over the usage of chemical preservatives in food processing [3] and their focus on minimally processed green-labeled foods is driving the food industry towards lactic acid bacterium based food preservation.
Biopreservation is the use of lactic acid bacteria (LAB) and/or their antimicrobial compounds to prevent spoilage and to extend the shelf life of foods.LAB has a long history of use as a bio-preservative and is "generally regarded as safe" (GRAS).LAB is Gram-positive, anaerobic but aero-tolerant, non-spore forming, non-motile, rod and coccus-shaped organisms.The preserving ability of LAB is mainly related to the production of organic acids and reduction of pH, which generally restricts growth of pathogenic bacteria and fungi [4].However, there are other metabolic products such as cyclic dipeptides and bacteriocins/antimicrobial peptides reported to involve in the preservation of many processed and natural foods [5].There are many reports on antifungal activities and possible application as biopreservatives of LAB from various sources [6] [7].
The GIT of chicken harbors a huge collection of micro flora more than 650 species, of which more than half are previously unknown bacterial genera [8].Lactobacilli from chicken origin are a good source of antimicrobial peptides [9].A very limited data are available in the literature on antibacterial and antifungal probiotic lactobacilli isolated from chicken GIT, which is known for harbouring highly diversified microbial species [10].Probiotics are defined as live microorganisms that when administered in adequate amounts, they confer a health benefit on the host [11].The principle requisite for selection of good probiotic includes product safety for human and animal consumption and survival in the GIT.
In the present study, we have isolated and characterized LAB from chicken GIT with strong and broad antibacterial and antifungal activity against pathogenic food spoilage microbes.The probiotic characteristics were also tested in vitro.

Isolates, Cultures and Growth Conditions
LAB was isolated from the crop, intestine, gizzard and ceca of seven weeks old broiler chicken.Serially diluted samples were inoculated onto acidified de Mann-Rogosa-Sharpe (MRS) agar (Himedia, India) and incubated anaerobically at 37˚C for 48 h.The MRS agar plates were acidified at pH 4.0 and supplemented with 0.2% (W/V) sodium azide to support the growth of LAB during isolation.After cultivation, creamy, opaque and elevated single colonies were picked and streaked on MRS agar.Gram positive, catalase negative isolates were selected and stored at -70˚C in MRS broth with 30% glycerol., Chandigarh, India.The target fungi were selected as representative of baked goods spoilage fungi and bacterial pathogens were selected representing frequent food contaminants.
Molds were cultivated on potato dextrose agar (PDA) (Himedia, India) slants at 30˚C for 6 days or till sporulation and stored at 4˚C.The spores were harvested from slants in sterile peptone water (0.2% W/V) to prepare the inoculum containing 1 × 10 5 spores per mL.Bacterial pathogens were cultured in tryptone-glucose-yeast extract (TGY) (Himedia, India) broth at 37˚C for 24 h and stored at -70˚C in TGY broth with 30% glycerol.E. coli DH5α was grown in Luria-Bertani (LB) medium at 37˚C and 180 rpm.E. coli transformants were cultivated in LB medium supplemented with ampicillin (50 µg/ml) (Hi media, India).Plasmid pTZ57R/T (Thermo Scientific, USA) was used for the construction of recombinant plasmid carrying the plantaricin EF gene.

Primary Screening of Isolates for Antifungal Activity
Bacterial isolates grown in 5 mL of MRS broth at 37˚C for 24 h were checked for their antifungal activity against target fungi by the overlay method described by Y.I.Hassan and L.B. Bullerman, 2008 [12] with minor modifications.A total of 10 µL of each LAB isolate were added as a discrete spots in the centre of buffered MRS agar plates and incubated anaerobically at 37˚C for 24 h.The medium was buffered at pH 6.5 by 75 m•mol•L −1 KH 2 PO 4 to avoid the effect of pH on antifungal activity.After incubation, the plates were overlaid with 15 mL soft PDA (0.8% agar) containing 1 × 10 5 spores per mL and incubated at 30˚C.The incubation period lasted up to 4 days and clear zones of inhibition around the bacterial spot were recorded and scored as follows: (−) no visible inhibition; (+) spore formation delayed with a small clear zone around the colony; (++) spore formation delayed with a good clear zone around the colony; (+++) extensive suppression of spore formation and mycelial growth with definite clear zone around the colony.The experiment was performed in triplicates and Lactobacillus isolates with very good antagonistic activity were selected for further studies.
Antifungal activity of LAB isolates on the growth of Aspergillus niger was investigated by dual agar plate assay or confrontation assay described by Y.I.Hassan and L.B. Bullerman, 2008 [12] with minor modification of using buffered MRS agar.MRS agar buffered at pH 6.5 was mixed with 100 µL of actively grown LAB culture at the bottom of a petri plate and incubated at 37˚C for 24 h.After incubation, the plates were overlaid with soft PDA and 10 µL of mold spore suspension was inoculated as a discrete spot on to the centre of each plate.Then the plates were incubated at 30˚C for 10 days.The diameters of the growing mold colonies were measured in control and test plates.These data were used to plot growth curves and to select potent antifungal LAB isolate.The nature of antifungal activity was investigated with 10-fold-concentrated culture filtrate treated with acid protease (2 mg•ml −1 ) (Sigma, India) in 10 mM citrate buffer pH 3.0 and incubated at 37˚C for 3 h (Magnusson and Schnürer, 2001).Before evaluating the antifungal activity, the pH of the supernatant was readjusted to pH value 5.0.Both 10-fold-concentrated MRS broth treated with enzyme and pH adjusted 10-fold-concentrated samples served as controls.

Screening of Isolates for Antibacterial Activity
Antibacterial activities of LAB isolates were determined against target bacterial pathogens by agar well diffusion method as described earlier [13].The bacteriocinogenic nature of antibacterial activity was determined by using 10-fold-concentrated CFS treated with acid protease (2 mg•ml −1 ) (Sigma, India) in 10 mM citrate buffer pH 3.0 and incubated at 37˚C for 3 h.Antibacterial activity was checked at pH 6.0 against Staphylococcus aureus and compared with control which was also processed in a similar way without enzyme [14].

Classical Characterization of Potent Antifungal and Antibacterial Isolates
Physiological and biochemical attributes of potent antifungal and antibacterial LAB isolates were determined by methods as described earlier [15].Sugar utilization profile of isolates was resolved by using the HiCarbo Kit (Himedia, India).The configurations of lactic acid enantiomers and their concentrations were determined by enzymatic method as described earlier [16] [17].
( ) 340 3 Lactate mM 6.22 vol of sample 1 where 3 is reaction volume, 6.22 is mM extinction coefficient of NADH at 340 nm and 1 is path length of cuvette in cm.

Antibiotic Susceptibility
Antibiotic susceptibility of isolates was assayed by the disc diffusion method.To determine antibiotic susceptibility, isolate was seeded in MRS agar (1% agar w/v) with 10 8 CFU/mL and allowed to get solidified.The commercial antibiotic discs of different concentrations were placed on the surface of the medium and incubated at 37˚C for 24 h.The resistance and sensitivity were noted as per CLSI/NCCLS standard [18].

Molecular Characterization of Isolates
Genomic DNA was extracted using the method of Anderson and Mckay [19].RAPD finger printing was performed using the primers M13 (5'-GAGGGTGGCGGTTCT-3') and R2 (5'-GGCGACCACTAG-3') as described earlier [13].The polymorphic DNA pattern was analyzed by running amplified products in 1.5% agarose gel electrophoresis with 500 bp DNA ladder (Sigma, USA).M13 RAPD profiles were visualized under UV light and analyzed with Quantity one software (Bio-Rad, USA).The similarities were calculated using the Dice coefficient.The dendrogram was constructed by means of the unweighted pair group method using arithmetic averages (UPGMA).
The potent isolates were identified by 16S ribosomal RNA (rRNA) sequencing after amplification using primers fKJ (5'-CATTGGGACTGAGACACTGC-3') and rKJ (5'-CACCGCGACATGCTGATTC-3') [17].The sequences were compared with reference sequences in Gen Bank at the National Centre for Biotechnology Information (NCBI) using a BLASTn program.Phylogenetic tree for isolates was constructed using the neighbor-joining method with MEGA 5.0 software.The 16S rRNA sequences of isolates were deposited in Gen Bank.

PCR Amplification and Cloning of Plantaricin EF Gene
PCR amplification of plantaricin gene EF (plnEF) was performed from genomic DNA of isolates using primers plnEF-F (5'-GGTGGTTTTAATCGGGGCGG-3') and plnEF-R (5'-ACTTGATGGCTTGAACTATCC-3') [21].50 µL of PCR mixture contained 0.5 µM of primer, 100 ng of genomic DNA, 1 X Taq DNA polymerase buffer, 1 U of Taq DNA polymerase, 0.2 mM of each dNTP and 1.5 mM MgCl 2 .PlnEF gene amplification was performed in a thermo cycler at 94˚C for 3 min, followed by 29 cycles of 1 min at 94˚C, 1 min at 58˚C and 2 min at 72˚C with an extension of 72˚C for 7 min.Amplified plantaricin EF gene was purified using a clean-up kit (Merck Bioscience, India) and ligated into pTZ57R/T cloning vector by TA cloning method.Ligation mixture was transformed into E. coli DH5α competent cells, using standard procedure described by Sambrook et al., 2001 [22].The recombinant plasmid was confirmed by restriction digestion by EcoR I and Hind III, as well as by sequencing.Sequencing was performed at Eurofins Genomics India Pvt Ltd, India and sequence was submitted to Gen Bank data base.

Auto-Aggregation, Co-Aggregation and Cell Surface Hydrophobicity
Aggregation between the cells of same strains (auto-aggregation) or between genetically different strains (coaggregation) is of considerable importance in the human gut where probiotics are to be active and such abilities favour bacterial maintenance in the gastrointestinal tract.The autoaggregation and coaggregation (with E. coli, Listeria monocytogenes) assays were performed according to Osmanagaoglu et al., 2010 [23].Isolates were grown in MRS broth for 18 h at 37˚C.Cells were harvested and washed twice with phosphate buffer saline (PBS) pH 7.0.The cells were suspended in PBS, pH 7.0 and adjusted to an optical density at A 600 to1.0.To three milliliters of cell suspension, equal volumes of n-hexadecane was added, vortexed for 2 min and allowed to stand for 30 min for phase separation.The aqueous phase was collected and absorbance was checked at 600 nm.The optical density readings were used to calculate the percentage of cell surface hydrophobicity of isolate adhering to solvent [23].

Growth at Different pH Values and Bile Concentrations
Isolates were grown in MRS broth adjusted to pH 2, 2.5, 3.5, 7.5 and 8.5 for 24 h at 37˚C.The assay was conducted in sterile flat-bottom 96-well microtitre plates.Each well was filled with 180 µl of the medium and inoculated with 20 µl of the overnight cultures obtained in MRS broth (O.D. at 600 nm is 0.2) at 37˚C.Optical density readings were recorded at 600 nm every hour for 8 h and after 24 h, while cultures grown in MRS broth pH 6.5 served as the control [24].The experiment was performed in triplicates.Similarly, isolates were grown in MRS broth containing 0.05%, 0.1%, 0.3% and 0.6% of oxgall for 24 h.Optical density readings were recorded at 600 nm every hour for 8 h and after 24 h.Isolates grown in MRS broth without oxgall served as control.

β-Galactosidase Activity
Isolates were grown in MRS-lac broth and cells were harvested by centrifugation at 8000 rpm for 10 min.The cells were washed with saline and suspended in the Z buffer (60 mM Na 2 HPO 4 , 40 mM NaH 2 PO 4 , 50 mM β-mercaptoethanol pH7.0) and processed as described by Vinderola and Reinheimer, 2003 [25].The suspended cells were permeabilized with toluene/acetone (1:9, v/v).The reaction mixture having 900 µl of Z buffer, 100 μl of permeabilized cells and 200 µl of ONPG (4 mg/mL) was incubated at 37˚C for 15 min.The reaction was stopped with the addition of 1% sodium carbonate and absorbance was measured at 550 nm and 420 nm.The enzymatic activity was measured in Miller units.

Hemolytic and Bile Salt Hydrolase Activity
For testing hemolytic activity, isolates were streaked on Columbia agar plates (Himedia, India), containing 5% (w/v) human blood, and incubated for 24 h at 37˚C.Blood agar plates were checked for signs of β-hemolysis (clear zones around the colonies), α-hemolysis (green-hued zones around the colonies) or γ-hemolysis (no zones around the colonies) [26].Escherichia coli MTCC 728 and Streptococcus pyogenes MTCC 442 were used as control for α-and β-hemolysis, respectively.Bile salt hydrolase (BSH) activity was assayed according to Lee et al., 2011 [27].Isolates were streaked on MRS agar plates supplemented with 0.5% (w/v) sodium salt of taurodeoxycholic acid and 0.37 g CaCl 2 /L.The BSH activity was semi-quantified by the precipitation zones around the colonies after incubating anaerobically at 37˚C for 24 h.

Isolation and Screening of Lactobacillus for Antibacterial and Antifungal Activity
Lactic acid bacteria were isolated from GIT of seven weeks old broiler chicken.A total of 113 Lactobacillus strains (47 from crop, 8 from gizzard, 27 from intestine and 31 from ceca) were isolated based on their ability to grow on acidified MRS agar medium.All 113 isolates were screened for antibacterial activity against various LAB and food borne pathogens.All isolates showed activity against tested LAB and bacterial pathogens.Approximately 53% (60 isolates) of the total isolates showed good antibacterial activity against bacterial pathogens L. monocytogenes, S. aureus and E. coli, indicating that Lactobacillus isolates from chicken origin are potential candidates for producing antibacterial metabolites.These isolates were further screened for antifungal activity against filamentous fungi Aspergillus niger and Penicillium expansum.Among the tested spoilage fungi, Aspergillus niger growth was inhibited by a dominant number of isolates, whereas Penicillium expansum growth was affected by few isolates.A total of six bacilli isolates VJC6, VJC11, VJC13, VJC23, VJI16 and VJI17 having similar morphology (Figure 1) were chosen for further study, based on their capability to inhibit all tested bacterial pathogens and food spoilage fungi (Figure 2 & Figure 3, Table 1 & Table 2).All the six isolates were found to be bacteriocinogenic in nature as they lost antibacterial activity upon treatment with acid protease [14] [28] (Figure 2(a), while the antifungal activity persisted indicating a non-proteinaceous substance was involved (Figure 3(b)).In the earlier study by Y.I Hassan and L.B. Bullerman, 2008 [12], it was reported that Aspergillus species were the most resistant to antifungal activity and difficult to inhibit by LAB.In our study, all the six isolates showed complete inhibition of Aspergillus niger for 7 days and continued to show same even for 3 months, indicated their preserving ability against fungal spoilage (Figure S1).These isolates were identified by 16S rRNA   The following scale was used: (-) no visible inhibition; (+) spore formation delayed with a small clear zone around the colony; (++) spore formation delayed with a good clear zone around the colony; (+++) extensive suppression of spore formation and mycelial growth with definite clear zone around the colony.
gene sequence and multiplex PCR analysis.The genomic variability of six isolates was studied by RAPD PCR analysis.

Biochemical and Physiological Characterization
All the isolates were Gram-positive, catalase-negative, rod shaped, aerobic or micro-aerobic, homo fermentative bacteria, and produce D and L-lactic acid (10 -20 mM).These morphological characters suggested that all the isolates belonged to the genus Lactobacillus [29].All the Lactobacillus strains grew in wide ranges of NaCl concentrations (1% -6.5%), temperatures (15˚C -37˚C) and pH (4.5 -8.5), with optimum growth at pH 6.5.The biochemical tests revealed that all Lactobacillus strains were acetoin producers and unable to hydrolyze gelatin, starch and arginine.The carbon utilization pattern was checked for six Lactobacillus strains and they can ferment glucose, fructose, sucrose, mannose, lactose and trehalose, but not xylose, raffinose, glycerol, melibiose, sodium gluconate, erythritol, α-methyl-D-glucoside and α-methyl-D-mannoside.The sugar profiles of the six isolates were varied and broad (Table S1).Biochemical studies indicated that all the six isolates belonged to Lactobacillus genus.

Antibiotic Susceptibility
All the six isolates were assayed for their susceptibility to 22 antibiotics (Table S2).They were found resistant to inhibitors of nucleic acid synthesis (norfloxacin, nalidixic acid and ciprofloxacin), inhibitors of protein synthesis (amikacin and streptomycin), inhibitors of cell wall synthesis (cepharadine and cephaloridine), an inhibitor of cytoplasmic membrane function (colistin), urinary tract antiseptic (nitrofurantoin) and sensitive to chloramphenicol, tetracycline, erythromycin, lincomycin, mecillinam, ampicillin, penicillin-G, and co-trimoxazole.The observed antibiotic resistance and sensitivity are in accordance with the findings of Mathur and Singh, 1995 [30].

Genotypic Characterization by RAPD-PCR
To exclude the clonal relatedness among the six isolates, RAPD analysis was performed using two primers M13 and R2.The reproducibility of the RAPD analysis was assessed by comparing the PCR products obtained from two separate cultures of the same strain for all the six isolates.Among two primers, M13 was the most useful for distinguishing six Lactobacillus strains, producing a variant DNA banding pattern for all the six isolates (Figure S2).The UPGMA dendrogram analysis revealed that the six Lactobacillus strains were genetically different at strain level and there was a more genetic distance at the strain level between VJI17, VJC23 and the rest of the isolates (Figure S3).

Comparative 16S rRNA Sequence Analysis and Multiplex PCR Assay
The 16S rRNA gene of the six isolates was sequenced and analysed for similarities using NCBI BLAST search program.It showed that all the six isolates had high sequence similarity (100%) to Lactobacillus plantarum (AF1), Lactobacillus paraplantarum (N957) and Lactobacillus pentosus (MH53).As these three species are genotypically closely related (>97%), 16S ribosomal DNA sequences of the isolates are not suitable for species identification.The phylogenetic tree was constructed using the neighbor-joining method (Figure 4).The presumed six Lactobacillus strains were further confirmed at the species level by multiplex PCR.Multiplex PCR assay based on recA gene gave the amplification product of 318 bp for all the six isolates belonging to Lactobacillus plantarum (Figure S4).The 16S rRNA analysis and multiplex PCR assay conferred that these isolates were belonged to Lactobacillus plantarum.

PCR Amplification and Cloning of Plantaricin EF Gene
The isolates were tested for the presence of known bacteriocin genes by PCR.Plantaricin EF specific primers plnEF-F and plnEF-R were used to amplify and sequence a 306 bp fragment from all the isolates (Figure S5).The nucleotide sequence showed 99% similarity to the plantaricin E and F genes on the genome of Lactobacillus plantarum strain C11 (Gen Bank: X94434.2).The sequence analysis by BLAST at NCBI with published sequences revealed that PCR amplified 306 bp fragment consisting of a partial plnE gene (1 -108 bp) and a complete plnF gene (133 -291) (Gen Bank: KM347970).The plnEF gene was cloned in to pTZ57R/T cloning vector by TA cloning method and transformed in to E. coli DH5α cells.Double digestion of the recombinant vector with EcoR I and Hind III enzymes, resulted in the release of 400 bp insert and vector fragment (Figure S6).
Plantaricin is a type IIb two-peptide bacteriocin obtained from different strains of Lactobacillus plantarum [31].These findings further confirmed that the isolates were belonged to Lactobacillus plantarum and bacteriocinogenic nature may be due to the expression of plnE and F genes.

Probiotic Properties of Lactobacillus
Autoaggregation and coaggregation are vital properties of a probiotic organism, responsible for biofilm formation on the gut epithelium.The binding of probiotic organism with intestinal pathogens will be helpful to kill pathogens by antimicrobial substances [32].All the isolates exhibited bacteriocinogenic mode antibacterial activity.The tested isolates exhibited strong autoaggregating phenotype ranged from 78% -86%, which assist in binding firmly with intestinal epithelium.The Lactobacillus strains showed the most coaggregation ability with E.coli MTCC 728 (49% -61%) when compared to L. monocytogenes MTCC 657 (30% -46%).The resistance to harsh conditions of the stomach and intestine is another essential prerequisite for probiotic organism.The physiological pH of the stomach is 2 -3 and bile concentration is 0.3% [33].A good probiotic must be resistant to the acidic nature of the stomach and the physiological bile concentration.The isolates were remained unaffected by the acidic pH 2.0 -3.5 and good growth was observed at pH 6.5 -8.5 (Figure 5).They also showed resistance and growth in the presence of 0.6% of bile, indicating that these isolates can survive even at high  concentrations of bile (Figure 6).Bacterial cell hydrophobicity may assist in adhesion, but does not seem to be a prerequisite for colonization [34].The L. plantarum strains showed hydrophobicity ranging from 16% -33% with n-hexadecane.Lactose intolerance is caused by deficiency β-galactosidase in the lining of duodenum.So, the food products, fermented with lactase producers could help to treat lactose intolerance.All the isolates showed β-galactosidase activity.Among the isolates, VJC13 showed maximum β-galactosidase activity (1179 MU) may have application in the dairy industry.In this study no isolate showed hemolytic activity where as positive strains Escherichia coli MTCC 728 and Streptococcus pyogenes MTCC 442 showed α-and β-hemolysis, respectively.Table 3 gives the probiotic properties of the all the Lactobacillus strains.

Conclusion
In conclusion, chicken GIT is a good reservoir of potential antifungal, antibacterial and probiotic Lactobacillus plantarum strains with high diversity.The study of antifungal nature and probiotic properties in vivo of these isolates may lead to the development of lactic acid bacteria based preservative systems to prevent fungal spoilage in food and feed industries.
-galactosidase activity MU 1000 Time of incubation Volume of culture

Figure S5 .
Figure S5.PCR amplification of plnEF gene of six potent strains.

Figure S6 .
Figure S6.EcoR I and Hind III digestion of recombinant vector pTZ57R/T with plnEF gene. 1) EcoR I and Hind III double digest product, 2) 1 kb DNA ladder.

Table 1 .
Antimicrobial activity against LAB and various pathogens.

Table 2 .
Antifungal inhibition spectrum of Lactobacillus isolates in agar over layer method.