Anaerobe 27 (2014) 87e95

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Clinical microbiology

Antimicrobial potential of bacteriocin producing Lysinibacillus jx416856 against foodborne bacterial and fungal pathogens, isolated from fruits and vegetable waste Varish Ahmad a, A.N. Muhammad Zafar Iqbal b, Mohd Haseeb a, Mohd Sajid Khan a, * a b

Nanomedicine & Nanobiotechnology Lab, Department of Biosciences, Integral University, Kursi Road, Dasauli, Lucknow 226026, India Department of Studies in Zoology, Government First Grade UG & PG College, Karwar 581301, India

a r t i c l e i n f o

a b s t r a c t

Article history: Received 3 January 2014 Received in revised form 25 March 2014 Accepted 2 April 2014 Available online 13 April 2014

In this study, antimicrobial potential, some probiotics properties and bacteriocin nature of Lysinibacillus, isolated from fruits and vegetable waste were evaluated. For this, 125 Lactobacillus isolates were tested against foodborne bacterial and fungal pathogens. Among these, an isolated Bacillus spp. showed significant aggregation-co-aggregation probiotics properties and potentially inhibits the foodborne gram positive microbial pathogens such as Staphylococcus aureus, (22 mm ZOI), Staphylococcus epedermidis and Bacillus cereus (18 mm). Phenotypically and molecularly it was identified as Lysinibacillus (NCBI accession no. JX416856) and it was found closest to Lysinibacillus fusiformis, Lysinibacillus sphaericus and Lysinibacillus xylanilyticus. Physico-biochemically, it was found to be negative for amylase, protease, gelatinase, nitrate reductase and urease while positive for catalase. The diagnostic fatty acid was 22;2 (3.51). The growth conditions and bacteriocin activity were found to be optimum with MRS media at pH 7e10, Temperature 35e40  C and salt tolerance at 1e3%. Eventually its production was optimized with MRS broth at pH 7.6, 37  C, for 36 h in shaking conditions at the rate of 100 rpm. Active bacteriocin was isolated at 60% ammonium sulfate precipitation. The molecular weight of given bacteriocin was found to be nearly 25e35 kDa by SDS-PAGE. Based on physico- biochemical properties, the isolated bacteriocin was to be categories in class II bacteriocin. The bacteriocin was found to be stable in the range of 4e80  C temperature, 6e10 pH and even in the presence of surfactant (such as SDS and Tween 80). However, proteases like pepsin and trypsin were found to degrade the bacteriocin. Collectively, the broad spectrum inhibitory potential and physical stability offered the antimicrobial potential to Lysinibacillus, and its relevant bacteriocin might be used as an alternative food preservative or therapeutic agent to control spoilage of different food products. Ó 2014 Elsevier Ltd. All rights reserved.

Keywords: Lysinibacillus Bacteriocin 16S r RNA gene Phylogenetic analysis

1. Introduction In industrialized countries, nearly 30% population is infected with foodborne bacterial and fungal pathogens. In 2000 at least 2 million people even died from diarrheal diseases worldwide [1]. As a result, research has been accelerated to inhibit foodborne pathogens by safe preservative like essential oils, surfactants and bacteriocin particularly from lactic acid bacteria [2]. Lactobacilli inhibited other organisms by producing signaling and protective

* Corresponding author. Nanomedicine & Nanobiotechnology Lab, Department of Biosciences, Integral University, Lucknow, Uttar Pradesh 226026, India. Tel.: þ91 983 9179013; fax: þ91 522 2890809. E-mail addresses: [email protected], [email protected] (M. S. Khan). http://dx.doi.org/10.1016/j.anaerobe.2014.04.001 1075-9964/Ó 2014 Elsevier Ltd. All rights reserved.

molecules such as organic acids, hydrogen peroxide, CO2, diacetyl, acetaldehyde, D-isomers of amino acids, reuterin and bacteriocins [3e6]. Bacteriocin from lactic acid bacteria have been studied extensively as therapeutic and food preservative agents which was found very effective at nano molar concentrations against different bacterial pathogens [7e13]. Bacteriocin is synthesized by the ribosome and functionally characterized by molecular size, mode of action, biosynthetic route, self protection mechanisms and antimicrobial potential [14]. Previously, Most of the bacteriocins are described into four major classes, Class -1 bacteriocins or lantibiotics or extensively post translationally modified bacteriocins. The class II bacteriocins are small (10kd), non lanthionine, heat stable unmodified peptides that do not require post-translational modification for antimicrobial activity, class III bacteriocins are large (>30 kDa), heat labile proteins. However, class IV bacteriocins

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are complex bacteriocins, containing lipid or carbohydrate moieties. The class II bacteriocins are further subdivided into Listeriaactive peptides with the N-terminal consensus sequence of YGNGVXC (class IIa) [15e17]. Bacteriocin kills microbes through adsorption and translocation and induces leakage of ions through pores from cell membranes [18,19]. In recent years, a number of analytical and molecular techniques have been routinely used for the identification and characterization of microorganisms. More recently, use of 16S rRNA has also been proposed for the identification of unknown bacteria. The highly conserved 16S rRNA gene sequences are amplified by PCR and the presence of some variable zones in amplified sequence is used for identification purposes [20,21]. Lysinibacillus is a Gram-positive, rod-shaped, and round-spore forming bacterium in the family Bacillaceae of phylum fermicutes. Lysinibacillus is commonly found in soil, plants and animals [22]. The genome of Lysinibacillus sphaericus strain C3-41 was the first strain in the genus Lysinibacillus which is taxonomically classified on the basis of polyphasic cell wall peptidoglycan [23]. The biotechnological potential of many Bacillus also has been explored [24] but antimicrobial potential of Lysinibacillus is poorly reported. The present study focuses on the isolation and identification of bacteriocin producing isolates. However, evaluation of some probiotics properties and antimicrobial potential of isolated Lysinibacillus against foodborne microbial pathogens were also done. For this, phenotypic, molecular identification of strain JX416856 and subsequently, the characterization of antimicrobial properties of its relevant bacteriocin were carried out. Referring to the available literature, this is the first study reporting the bacteriocin production and antimicrobial potential of Lysinibacillus isolated from fruits and vegetable waste. 2. Materials and methods 2.1. Isolation of microorganism The selected isolates for this study were isolated from spoiled fruits and vegetable waste, collected from the local vegetable market, Lucknow U.P., India. The isolation was done on MRS (de Man, Rogosa and Sharpe-Hi-Media, India) agar medium seeded with serially diluted spoiled food sample and incubated at 37  C for 24 h. The colonies were randomly selected, screened for bacteriocin activity and stored at 20  C in 30% glycerol. 2.2. Microbial indicator strains Gram positive and Gram negative bacterial indicator strains were used to evaluate the antimicrobial activity of isolates. These were Bacillus pumilis (MTCC160), Bacillus subtilis (MTCC441), Staphylococcus epidermidis (ATCC12228), Salmonella abony (NCTC6017), Pseudomonas aeruginosa (ATCC9027), Staphylococcus aureus (ATCC6538), Vibrio cholera (ATCC 14033), Micrococcus luteus (10240), Listeria monocytogenes (ATCC19115), Bacillus cereus (ATCC 14579) and Raoultella planticola (MTCC530). Lysinibacillus isolate was also tested against fungal strains Aspergillus niger (ATCC4695), Aspergillus flavus (ATCC 16872), Aspergillus fumigates (ATCC1022) Fusarium oxysporum (ATCC 1198) and Trichoderma viridae (52438). The microbial strains were obtained from the NCIM (National Collection of Industrial Microorganisms), National Chemical Laboratory (NCL) Pune, India. Bacterial strains were maintained at 20  C in 30% glycerol and sub cultured on nutrient agar (Hi-Media, India) while fungal strains were maintained on PDA (Potato dextrose agar, Merck, India) slants.

2.3. Assay for bacteriocin activity against bacterial and fungal indicators The preliminary inhibitory activity of each of the 125 isolates was determined by spot-On-lawn assay [25] against used indicators which was further confirmed by agar well diffusion assay [26]. Nutrient agar plates were inoculated with 100 ml (1  108 cfu/ml) of each bacterial indicator strain. A 100 ml filter sterilized (0.20 mm), neutralized, catalase (SigmaeAldrich) treated (2600 U/ml) cell-free supernatant (CFS) of bacteriocin producing isolate grown in MRS broth at 37  C for 36 h was filled into wells of agar plates (6 mm in diameter). Plates were incubated at 37  C for 24 h and antimicrobial activity was determined by measuring the diameter of inhibition zone around the wells. Antimicrobial activity was expressed as arbitrary units (AU) per ml. One AU is defined as the reciprocal value of the highest dilution showing a clear zone or growth inhibition [27]. Antifungal activity was evaluated with Lysinibacillus isolates by dual culturing as well as broth dilution method and percent inhibition was calculated [28]. Fungal cultures were inoculated on one side of the PDA medium and another side streaking of bacterial strain to be tested was done at approximately 3.5 cm from the fungal margin and incubated at the 25  C for seven days and percent inhibition against control was recorded [29]. 2.4. Evaluation of probiotics properties of Lysinibacillus Probiotics properties were evaluated spectrophotometrically, by calculating the auto aggregation, co-aggregation abilities and tolerance of different pH environments. Lysinibacillus culture and sensitive and resistant bacterial indicators were grown for 24 h, washed and resuspended in sterile saline solution (0.85% NaCl w/v) to OD590 ¼ 0.3. After 1 h incubation at 37  C, Lysinibacillus and Lysinibacillus with indictors strains were centrifuged at 300 g for 2 min at 20  C. The Aggregation abilities of Lysinibacillus was calculated using the following formula: % aggregations ¼ [(OD0 e OD60)]  100, where OD0 refer to initial OD and OD60 refer to the OD determined after 1 h of incubation [30]. Lysinibacillus (OD 0.3) was inoculated and incubated for 12 h at 37  C in MRS broth adjusted to pH 3.0, 4.0, 5.0, 6.0, 7.0 11.0, and 12.0 with 0.1 HCl and 0.1 NaOH. The pH tolerance of Lysinibacillus was determined by optical density, recorded at every hour for 12 h. Similarly, the ability of isolated Lysinibacillus to grow in the presence of different concentrations of bile was analyzed. Strain JX 416856 was incubated for 12 h at 37  C in MRS broth containing 0.2, 0.4, 0.6, 0.8, 1.0, 2.0 and 3.0% oxbile (SigmaeAldrich). Lysinibacillus grown in MRS broth without bile used as control and the entire test are performed in triplicates. 2.5. Kinetics of bacteriocin production Ten ml MRS broth culture of type strain Lysinibacillus JX416856 was incubated for 24 h at 37 C to produce mother culture for bacteriocin production by submerged fermentation. It was seeded to 90 ml previously autoclaved MRS broth followed by incubation. Samples were drawn after every 1 h and their absorbance (Abs590, Bio-Rad) was analyzed for change in biomass and bacteriocin activity (AU/ml). 2.6. Effect of bacteriocin containing CFS on the growth of indicator strain The growth inhibition of most sensitive bacterial indicator, Staphylococcus aureus was also analyzed by inoculating 10 ml filtersterilized, neutralized CFS in to 90 ml, log phase broth culture of S. aureus (4-h old culture Abs590 1.2) as an indicator bacterial strain.

V. Ahmad et al. / Anaerobe 27 (2014) 87e95

Inhibition was tested by observing O.D. at each 2 h during 12 h and as the final point after 24 h along with the control (MRS broth treated only). All the analysis was performed in triplicates. 2.7. Bacteriocin adsorption Bacteriocin adsorption on producer cells [31]. Briefly, Lysinibacillus (2%, v/v) was grown in 50 ml of MRS broth at 37  C for 36 h and the pH of the culture was adjusted to 7.0. After incubation Cells were harvested (8000 g, 15 min, 4  C) and washed with 0.1 M sterile phosphate buffer solution (pH 7). Afterwards, cells were resuspended in 10 ml of a solution of 100 mM of NaCl, stirred during 1 h at 4  C and centrifuged again (8000 g, 15 min, 4  C). The supernatant obtained was neutralized to pH 7.0 and tested for activity using the agar spot-test. Similarly, adsorption on indicator cells was conducted according to Todorov et al. [30] using recovered 60% ammonium sulfate precipitated active fraction of bacteriocin. The activity of unbound bacteriocin AU/ml was calculated by using the formula, % of bacteriocin adsorption ¼ [(unbound bacteriocin activity after treatment/original bacteriocin activity)]  100. Experiment was performed in triplicates.

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16S rRNA gene amplification by Polymerase Chain reactions using thermal cycler (BioradeThermal cycler). The used universal oligonucleotide primers 16SF (50 -AGAGTTTGATCCTGGCTCAG-30 ) and 16SR (50 - GGTTACCTTGTTACGACTT-30 ) were purchased from Ocimum Biosolution Hyderabad, India. The program used to amplify the 16S rRNA gene (BioradeThermal cycler) was- preheating at 95  C for 5 min of 1 cycle, followed by 30 cycles of 94  C for 1 min, 55  C for 1 min, and 72  C for 1 min. After these cycles, the reaction was maintained at 72  C for 10 min and then cooled to 4  C. 5 ml of the PCR products was analyzed on 1% agarose gel using 2 Kb molecular weight markers. The Big Dye Chemistry method was utilized to sequence the amplified gene as per the manufacturer’s protocols (Applied Biosystems 3730 XL DNA Analyzer, Ocimum Biosolution Hyderabad, India). Softwares used for sequence alignment and comparisons were CLUSTAL X (Version1.8msw; and ChromasPro1.7.5 Sequencing Analysis software. The evolutionary history was inferred from 1000 replicates using the NeighborJoining method [36]. 3. Results and discussion 3.1. Isolation and antibacterialeantifungal activity

2.8. Isolation and partial characterization of bacteriocin Bacteriocin containing CFS of Lysinibacillus was produced as described previously. However, bacteriocin was primarily extracted in butanol, air dried and reconstituted in DMSO. DMSO reconstitution of butanol extracted fraction was further tested for antimicrobial activity against indicator strains [32]. However, proteinaceous nature of bacteriocin in CFS and butanol extracted fraction were qualitatively confirmed by Biuret, Ninhydrin and Lowry test. Thin layer chromatography of butanol extracted fraction was also performed using butanol, acetic acid and water (4:1:1) as solvent, bands were visualized by 0.2% ninhydrin in acetone and Rf value was calculated. 20%, 40%, 60% and 80% Ammonium sulfate precipitated fraction of bacteriocin was also tested for antimicrobial activity. The bacteriocin from 60% ammonium sulfate fractions were extracted [33] which were further confirmed by SDS-PAGE using 5% stacking gel and 12% resolving gel. The inhibitory characteristics of isolated bacteriocin like effect of pH, temperature, surfactant and proteolytic enzymes against indicator strain was also tested [27]. 2.9. Taxonomic identifications Isolate JX416856 was identified by phenotypic characterization and molecular characterization. Morphology, staining, enzymatic profile, drug sensitivity and carbohydrate fermentation were analyzed [22,34]. For fatty acid analysis, extraction was done according to Sasser (1990) [35] and analyzed by Advanced Instrumentation Research Facility (AIRF) Jawaharlal Nehru University, New Delhi India, with gas chromatography using FAME mix 2 mg ml. The cellular fatty acids were identified by equivalent chain length of each compound to a peak naming that contains over known standard [22]. Peptidoglycan and isoprenoides quinines were analyzed by utilizing microbial identification services of Royal Life Sciences, Hyderabad, India using GC and HPLC. Antibiotic susceptibility testing was performed using disc of streptomycin (50 mg), penicillin G (20U), chloramphenicol (100 mg), ampicillin (10 mg), gentamycin (30 mg), tetracycline (30 mg), (kanamycin 30 mg), neomycin (30 mg). Ofloxacin (30 mg), ciprofloxacin (30 mg), vancomycin (30 mg), cephalosporin (30 mg), doxycycline (30 mg) and norfloxacin (30 mg) Kirbey-Baurer disc diffusion method. 1.5 ml overnight grown MRS broth culture was used to extract DNA (Sambrook and Russell, 2001). The purified DNA was used for

In this study, 125 MRS grown bacterial isolates from fruits and vegetable waste, were screened for inhibitory effect against bacterial and fungal indicators. Nearly 10% were found significant inhibitors of pathogenic bacteria and fungi. One isolate JX416856 strongly inhibited foodborne bacterial and fungal pathogens which was aerobic, gram positive, rod in shape and found to be as Lysinibacillus on the basis of phenotypic and molecular studies. Lysinibacilli are Gram-positive rods, endospore forming, aerobic bacterium in the family Bacillaceae of phylum fermicutes and commonly found in soil, plants and animals like puffer fish liver specimens [22]. Since isolate JX416856, strongly inhibited gram positive and gram negative bacteria (Table 1 & Fig. 1), therefore, this strain was selected for further studies. Gram positive indicators used in this study were Bacillus pumilis, Bacillus subtilis, Bacillus cereus, S. aureus Listeria monocytogenes and gram negative S. abony, R. planticola, Pseudomonas aeruginosa, Vibrio cholera and Mycobacterium luteus. Based on antimicrobial activity, isolate was found to be most active against gram positive S. aureus (22 mm ZOI) followed by B. cereus and S. epidermidis (18 mm ZOI) (Table 1). The antimicrobial activity of some food isolates due to bacteriocin has already been described [33]. The fungal inhibition tested by dual culturing and broth dilution methods showed significant inhibition of all the fungal indicators used in this study (Table 1 Fig. 1b,c). It was analyzed from the broth dilution study that Aspergillus flavus and Aspergillus fumigatus inhibited up to 93% followed by F. oxysporum (91%), Aspergillus niger 82% and Trichoderma viridae 76% (Table 1). Through broth dilution method it was found that CFS of isolated strain was found to be significantly effective against tested fungal strains and percent fungal inhibitions also found to be increased with increase in the concentration of CFS. The authors proposed that this phenomenon was due to either high concentration or presence of potential inhibitory molecule in the CFS. Many researches about the antifungal activities of bacillus showed that inhibitory compound of different nature could be involved [37,38] but the antimicrobial results analyzed (Table 1) from protease, catalase treated, neutralized CFS, indicted that inhibitory molecule was found to be bacteriocin, which was confirmed by TLC, ammonium sulfate precipitation and SDS-PAGE analysis of active fraction. The antifungal nature of CFS of Bacillus was described by Ahmadova et al. (2013) [33] and Voulgari et al. (2010) [39]. The bacteriocin of this isolate was found to be significantly effective against bacterial and

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Table 1 Antimicrobial effects of bacteriocin producing Lysinibacillus JX416856. Isolate fraction

Zone of inhibition (mm)

Bact

B.pum

B.sub

S.epi

S.abo

R.pl

S.aur

V.cho

M.lut

B.cer

P.aer

L.mon

CFS D*

R R

14  0.5 15  0.5

18  1 17  1

R R

R R

22  0.5 20  0.5

18  0.5 16  0.5

R R

18  1 16  1

10  1 10  1

R R

Antifungal effect of bacteriocin producing Lysinibacillus JX416856 A. flavus (þþþ) 93%

Fungal strains D-C % Inhibition by CFS

A. fumigates (þþþ) 93%

A. niger (þþþ) 82%

TLC

Rf (mm) value of bacteriocin bands in butanolic extraction

SPOTS

41

57

62

F. oxy (þþþ) 93%

82

T. viridae (þþþ) 76%

87

91

CFS Cell free supernatant; D*MSO reconstitute; D-C* dual culturing effect; (þþþ) strong inhibition (>70%).

fungal pathogens but larvicidial activity of purified bacteriocin of this strain need to be evaluated. 3.2. Probiotic properties Lysinibacillus JX416856 were found to show a significant level of auto aggregation (62.81%). Co-aggregation, which found to be different for each tested pathogenic strains of B. cereus (77.8%), B. pumilis (58.65%), S. aureus (93.64%) (Fig. 2). This might be due to different physicochemical characteristics of the cell surface [40,41]. These properties of studied strain could be beneficial because aggregation and co-aggregations with pathogens are the prerequisite to a probiotics for colonization and constitution of host defense mechanism against infection of gastrointestinal tract. High growth of Lysinibaciilus was to be shown in MRS broth with pH range of 5e9 (Abs590 3.852). Further decreased (Abs590 1.621) in growth was found with increase or decrease in pH value from above observed range of pH. This indicated that isolated Lysinibacillus could optimally grown at neutral pH but slightly increase in Abs590 from the inoculated, represented that studied isolate is acid resistant (Fig. 3). Resistance to bile salts give an ideal choice for the selection of probiotic bacteria and bacterial tolerate the 0.15e0.3 concentration Of bile 0.15e0.3% of bile are good probiotic bacteria for human use [42]. The strain JX 416856 was observed to be resistant only at concentrations of oxbile of 1.2%. Higher concentrations of oxbile affected the growth of isolate. 3.3. Inhibition kinetics The screening results indicated that gram positive bacteria found to be most sensitive against antimicrobial compound followed by gram negative bacteria. Therefore, gram positive S. aureus was selected as an indicator strain for further evaluation of the

compound. The antimicrobial effect started after 15 h of incubation and growth dependent bacteriocin activity was observed at early log phase (18 h), which increased slightly and remained constant to stationary phase (36 h) and eventually, decreased with a constant level (Fig. 4). During the growth a slight but constant (pH 6) change from the mother culture (pH7.6) was observed indicating slight acid production. Similarly, the effects of incubated temperatures are shown in Fig. 5. In kinetic studies, it was analyzed that the production and inhibition of bacteriocin was time, temperature and pH dependent which was found to be optimized with MRS broth at pH 7.6, 37  C, for 36 h of incubation in shaking conditions at 100 rpm. The optimum activity of bacteriocin was found to be in the range of pH 7e10, nearly 98.23% residual bacteriocin activity was analyzed within this range but the activity was found to reduce in the pH range of 10 (Table 2). Similarly, the residual activity between 10  Ce80  C was found to be 97.85% while above 80  C no bacteriocin activity was observed. The stability of bacteriocin at different pH and Temperature have been described by Mataragas et al., 2002, Casla et al., 1996 [43,44]. The decrease in activity might be due to degradation or aggregation [45] but alkaline bacteriocin from lactobacilli has also been described [31,46]. 3.4. Physico-biochemicals characterization of bacteriocin The neutralized, catalase treated CFS showed increased activities when it was treated with SDS and EDTA. Moderate heat stability was to be observed up to 80  C but lost completely when exposed at temperature >80  C and proteolytic enzymes pepsin and trypsin. The effects of physicochemical parameters on bacteriocin activity are shown in Table 2. These features indicated the protein nature of inhibitory molecules (bacteriocin) which was further confirmed in the next study of isolation, extraction and characterization. The adsorption of bacteriocin to the sensitive

Fig. 1. Antimicrobial effect of Lysinibacillus isolates JX416856 against bacteria and fungi. a: activity of Lysinibacillus against bacterial indicator S.aureus, b: activity of isolated JX416856T against Aspergillus flavus by dual culturing methods, c: activity against Aspergillus niger by broth dilution method.

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Fig. 2. Auto-aggregation and co- aggregation of Lysinibacillus jx416856 isolate with resistant strain of B. pumilis and sensitive strains of B. cereus and S. aureus.

strains is a first step to find out mode of action of any bacteriocin. Producer cells were not found to adsorb bacteriocin of their own because no bacteriocin activity (zero AU/ml) was observed in supernatant obtained by their washing. However, partially purified bacteriocin of Lysinibacillus was absorbed more by sensitive cells (742 AU/ml) than resistant cells (300 AU/ml) of indicator strains. A sharp decrease in OD590 (>80%) of bacteriocin containing indicator bacterial cells, was analyzed which might be due the bactericidal nature of Lysinibacillus bacteriocin. The activity was retained up to 80  C, indicated it moderate heat stable nature (Table 2). Various heat stable bacteriocin of class II has been described previously [26,45e49]. Activity with EDTA alone or with proteases has been found to be increased due to chelating of divalent ions from the protective outer cell membrane of bacterial indicator strain and inactivation of protease (Table 2) [50]. Similarly, SDS exposed the folded surface affecting the native structure of bacteriocin (Table 2). A significant antimicrobial profile from butanol extracted, DMSO reconstituted fraction indicated its stable nature in organic solvents which also supported the proteinaceous

Fig. 3. Growth and bacteriocin production of Lysinibacillus jx416856 at different pH values. Optimum production of bacteriocin was achieved at the pH 7e8.

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Fig. 4. Production of bacteriocin at different time intervals. Optimum production of bacteriocin was achieved at the time incubation of 30e36 h.

nature and solubility [1]. In SDS-PAGE analysis of active fraction, an active band of nearly 25e35 kDa of protein was observed. Thus, physicochemical properties like stability at pH, heat, organic solvent, antimicrobial profile and molecular weight support the notion that this bacteriocin might belong to class II bacteriocin which strongly inhibit the gram positive and gram negative bacterial pathogens [3,7,48]. Bacteriocin OR-7, enterocin E50-52, and enterocin E760, have been shown also to be active against both Gram-negative and Gram-positive bacteria, including Campylobacter jejuni, Yersinia sp., Salmonella sp., Escherichia coli O157:H7, Shigella dysenteriae, S. aureus, and Listeria sp. [7,46,48,51,52]. 3.5. Bacteriocin extraction and partial characterization The proteinaceous nature of the Protease sensitive active compound was further tested by extracting with butanol. DMSO reconstitute of butanol extracted fraction was found to be similarly effective (20 mm ZOI) as inhibition was observed with CFS. Various organic solvents like butanol and chloroform have been used to extract bacteriocin [53,54]. Proteinaceous nature of bacteriocin in butanol extraction, was further confirmed by TLC (6 bands) and qualitatively by ninhydrin and Biuret test. The calculated Rf values

Fig. 5. Production of bacteriocin at different Temperature. Optimum production of bacteriocin was achieved at the incubation of 30e37  C.

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V. Ahmad et al. / Anaerobe 27 (2014) 87e95 Table 2 Effect of physico-chemicals treatment on the antimicrobial activity of cell free supernatant of Lysinibacillus JX416865. Treatment

Residual activity (%)a

Temperature 4  Ce20  C 10  Ce80  C >80  Ce120  C pH (2e5) pH (6e10) pH (12) Pepsin Trypsin Butanol Ethyl acetate ETDA-25 mM ETDA-50 mM NaCl e 200 mM NaCl e 400 mM NaCl e 600 mM SDS (1%) Tween 80 (1%)

98.6  0.6 97.85  0.09 0 0 98.23  0.14 0 0 0 97.68  0.23 94.34  0.5 76.98  0.08 97.88  0.06 75.14  0.45 83.21  0.05 23.65  0.5 99.2  0.02 94.86  0.02

a Results are presented as the mean value of triplicate trials () standard deviation (SD).

of these bands were found to be 41, 57, 62, 82, 87 and 91 mm. The active fraction of bacteriocin was recovered with 60% ammonium sulphate saturation. 12% SDS-PAGE analysis of this fraction showed five major bands of proteins of molecular weight in the range of 25e70 kDa (Fig 6). The active bacteriocin bands were confirmed by overlay gel assay which was found to be of nearly 25e35 kDa protein.

Table 3 Physico-biochemical characteristics of Lysinibacillus JX416856. Morphological characters

Enzymatic characterization

Colony Morphology Gram stain Motility Endospore

Amylase Negative Protease Negative Catalase Positive Gelatinase Negative Urease Negative Nitrate reductase Negative MRVP Negative Carbohydrate fermentation Gelatin Negative Starch positive e no gas Maltose Positive e with gas D-glucose Positive e with gas Dextrin Negative e no gas D-glucose Positive e no gas Ribose Positive e no gas Lactose Positive e no gas Fructose Positive e no gas Sucrose Positive e no gas Manitol Positive e no gas

White round Bacillus Positive Motile Positive

Growth characters MRS Very good (þþþ) LB Very good (þþþ) GB Poor (þ) NaCl e 0.5% poor (þ) NaCl e 1% poor (þ) NaCl e 1.5% Very good (þþþ) NaCl e 3% Very good (þþþ) NaCl e 5% No growth () pH e 4 Poor (þ) pH e 7 Very good (þþþ) pH e 10 Very good (þþþ) pH e 12 No growth () T e 10  C Poor (þ) T e 20  C Moderate (þþ) T e 30  C Very good (þþþ)

Drug susceptibility of isolate JX416856 Drugs* ZOI

T 30

P R

E 22

AMP 20

OF 16

CIP 26

K 15

VAN R

CE 16

DOX R

NF R

Drugs* T ¼ Tetracycline, P ¼ Penicillin, E ¼ Erythromycin, AMP ¼ Ampicillin, OF ¼ Ofloxacin, CIP ¼ Ciprofloxacin, K ¼ Kanamycin, VAN ¼ Vancomycin, CE ¼ Cephalotoxime, DOX ¼ Doxycyclin, NF ¼ Nitrofurantoin.

3.6. Taxonomic identification of isolates Morphologically isolated cells of JX416856 were found to be gram positive Bacilli, with white creamish, round colonies of entire margin. Optimum growth of the isolate was observed in MRS medium and moderately was seen in LB broth followed by Nutrient broth. The 1.5%e3% NaCl salt concentration was found to be optimum. The optimum pH range was found to be 7e10 and optimum Temperature for growth was observed to be at 30e40  C. Further, it was found to be negative for enzymatic sensitivity with amylase, protease, gelatinase, nitrate reductase and urease but positive with catalase. The isolated cells were also characterized by drug

Table 4 Cellular fatty acid composition of Lysinibacillus JX416856Tand the type strains of species of genus Lysinibacillus.

Fig. 6. SDS-PAGE analysis of bacteriocin from ammonium sulphate precipitated fraction; Lane: M Molecular Marker; Bact : 60% Ammonium sulphate fraction of bacteriocin.

Fatty acids

1

2

3

4

5

14:0 ISO 14:0 15:0 ISO 15:0 ANTEISO 15:0 16:1 w7c alcohol 16:0 ISO 16:1 w11c 16:0 ISO 17:1 w10c 17:0 ISO 17:0 ANTEISO 18:n9c 20:0 21:0 22:2 Summed features 4

1.47 1.06 49.07 8.55 3.8 8.06 4.96 6.88 1.07 4.81 5.38 4.32 12.08 e e 3.51 3.68

1.58 e 30.29 1.82 e 16.24 25.59 4.25 2.7 4.43 10.59 1.6 e

1.87 1.01 49.01 9.25 e 7.93 5.51 2.2 e 5.87 7.22 4.81 e

e e 1

e e 3.21

1.25 e 48.58 9.27 e 8.29 5.15 3.54 1.02 5.92 4.7 3.38 e 6.7 e e 3.61

3.66 e 49.85 3.34 e 14.44 12.12 1.72 e 3.23 8.07 1.07 e e e e 1

*Strains: 1. Lysinibacillus JX416856; 2. L. pakiastanensis NCCP-54; 3. L. xylanilyticus KCTC13433; 4. L. fusiformis KCTC3454; 5. L. sphaericus KCTC3346.

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alcohol (8.06) and C15:0 ANTEISO (8.55). The specific acid observed was C21:0 (3.51%). The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol Phosphatidyl ehthanolamine and peptidoglycan was type A4a. This analysis also supported by the results of sequence similarity and phylogenetic tree analysis because the A4a peptidoglycan type is the key marker for the genus Lysinibacillus [34]. The major isoprenoid quinine of strain JX 416856 was menaquinone-7 (MK-7), which was found same as that similar to members of genus Lysinibacillus [51].

Fig. 7. Agarose gel electrophoresis of 16 S rRNA amplified genes from Lysinibacillus JX416856. Lane M 2 kb Ladder, Lane-1: 1396 bp amplified product of 16S rRNA gene. (Middle lane: 16S rRNA amplified product of another isolate which was not described in this study).

sensitivity test carried out by disc diffusion method using a disc of standard drugs. Lysinibacillus JX416856 was observed resistant to P, VAN, DOX and NF but found to be sensitive to fluroquinones CIP and OFL. The antibiogram and other phenotypical characteristics of isolate JX416856 are shown in Table 3. 3.6.1. Fatty acid methyl ester (FAME) analysis The cellular fatty acid methyl ester profile of type strain JX416856 is shown in Table 4 with reference to L. pakiastanensis NCCP-54; Lysinibacillus xylanilyticus KCTC13433; Lysinibacillus fusiformis KCTC3454 and L. sphaericus KCTC3346, (Hayat et al., 2013 [22]). The major fatty acids were C15:0 ISO (49.07%), C16:1 w7c

3.6.2. Molecular characterization and phylogenetic analysis of Lysinibacillus The 16S rRNA gene was amplified and given length was found to be 1396 bp (Fig. 7). Thus obtained assembled sequences were successfully submitted to NCBI with the accession number JX416856. Evolutionary analyses were conducted by Neighbour-Joining method using CLUSTAL 2.0.11. The obtained phylogenetic tree proved that it was found to be close to Lysinibacillus fusiformis (NR_42072) and Lysinibcillus boronotolereance bacterium (NR_41276) (Fig 8). A combination approach of phenotypic and molecular techniques was used for taxonomic identification of an isolate. 16Sr RNA gene (1396 bp) sequence similarity showed that it is closely related to Lysinibacillus. Isolate occupied a separate phylogenetic position in radiation of Lysinibacillus and other species of Bacilli. Further it was differ with some characteristics from previously described Lysinibacillus sp. [22,34] like unique 16SrRNA gene sequence, bacteriocin production, growth in MRS, presence of specific diagnostic fatty acid, all these features indicated that it is a novel species

Fig. 8. Neighbour-joining phylogenetic relationship of isolates JX416856 with closely related species of Lysinibacillus and other related genera. Bootstrap values Based on 1000 replication are shown at the branch nodes at the scale of 0.005.

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of Lysinibacillus (Tables 3,4). Intrinsic antibiotic resistance is also one of the characteristics that can differentiate groups of lactobacilli and other bacteria. Type strain JX416856 was found to be sensitive to T, E, AMP, OFL, CIP,K and CEPH and have been found to be resistant to P, VAN, DOX and NF. Previously described drug susceptibility analysis by Lee et al. (2013) [34] indicated that various Lysinibacillus species have shown characteristics drug susceptibility, differentiating among species. Lysinibacillus xylanilyticus sp. Nov.XDB9 was sensitive to novobiocin and streptomycin and resistant to chlorophenicol and gentamycin. L. boronni tolerance JCM21713 was found sensitive to novabiocin and gentamycin but resistant to chlorophenicol and streptomycin. Further L. fusiformis was sensitive to gentamycin and novobiocin but resistant to streptomycin and chlorophenicol. The type strain JX 416856 of this study showed different drug susceptibility from previously described Lysinibacillus sp. Thus characteristic drug susceptibility feature of this isolated strain again supported the novelty of this strain. This analysis is also indicating the diversity of drug susceptibility in Lysinibacillus sp., which represents different plasmid profile, helping isolates to survive in a different ecological niche. The optimized cultural characters, which were found to be similar to other Lysinibacillus as described by Lee et al. (2013) [34] (Table 3). It produced optimum bacteriocin at 37  C, at pH 7.6 in shaking conditions for 36 h further, it was found amylase, protease, gelatinase, nitrate reductase and urease insensitive while catalase sensitive (Table 3). The carbohydrates fermentation of starch, maltose, D-glucose, ribose, lactose, fructose, sucrose and mannitol were observed positive with or without gas formation (Table 3). The gas formation was observed only with maltose fermentation. The Lysinibacillus xylanilyticus sp. Nov.XDB9 was observed positive with asculin, arabinose, citrate, maltose, mellibiose, sorbitol, sucrose, xylose and negative with trehalose, ribose and mannose. Lysinibacillus fusiformis was found positive with ribose and citrate. Thus these observations indicated that each Lysinibacillus species also has a distinct and specific carbohydrate fermentation pattern. The differential fermentation pattern of L. xylanilyticus, L. fusiformis, L. boronnitolerance, and L. parviviboronnicapiens, supported the notion of being a novel species to type strain JX416856. Thus, based on conducted physicobiochemicals tests, molecular characteristics, a distinct phylogenetic position, presence of diagnostic fatty acid, specific antibiogram and bacteriocin production of this isolate support that it is a novel species of Lysnibacillus and could be designated as Lysinibacillus JX416856. 4. Conclusion To conclude, this is probably the first report on the bacteriocin production from Lysinibacillus, isolated from fruits and vegetable waste sample. In this study Lysinibacillus was isolated, purified, taxonomically identified and anticipated the potentiality of bacteriocin production from Lysinibacillus JX416856. The description of Lysinibacillus isolate was further concluded as gram positive, motile, endospore forming catalase positive rods. The colonies on MRS agar are white creamish, circular with entire margin. The growth conditions were optimized with MRS media at (pH 7e10, 7.4), (Temperature 30e40  C, 37  C) and salt tolerance at (1e3%, 2%). The strain was characterized biochemically as amylase, protease, nitrate reductase, gelatinase and urease insensitive. Thus presence of specific fatty acid methyl ester composition, carbohydrate fermentation pattern, drug susceptibility and bacteriocin production indicated the novelty of this isolate. The 60% ammonium sulphate fraction showed a nearly 25e35 kDa active band of bacteriocin. The bacteriocin of this isolate showed physicobiochemical stability at various physical parameters. Besides the broad spectrum inhibition and physical stability, additional

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Antimicrobial potential of bacteriocin producing Lysinibacillus jx416856 against foodborne bacterial and fungal pathogens, isolated from fruits and vegetable waste.

In this study, antimicrobial potential, some probiotics properties and bacteriocin nature of Lysinibacillus, isolated from fruits and vegetable waste ...
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