Bacteriocin PJ4 Active Against Enteric Pathogen ...

Bacteriocin PJ4 Active Against Enteric Pathogen Produced by Lactobacillus helveticus PJ4 Isolated from Gut Microflora of Wistar Rat (Rattus norvegicus): Partial Purification and Characterization of Bacteriocin Prasant Kumar Jena, Disha Trivedi, Harshita Chaudhary, Tapasa Kumar Sahoo, et al. Applied Biochemistry and Biotechnology Part A: Enzyme Engineering and Biotechnology ISSN 0273-2289 Volume 169 Number 7 Appl Biochem Biotechnol (2013) 169:2088-2100 DOI 10.1007/s12010-012-0044-7

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Author's personal copy Appl Biochem Biotechnol (2013) 169:2088–2100 DOI 10.1007/s12010-012-0044-7

Bacteriocin PJ4 Active Against Enteric Pathogen Produced by Lactobacillus helveticus PJ4 Isolated from Gut Microflora of Wistar Rat (Rattus norvegicus): Partial Purification and Characterization of Bacteriocin Prasant Kumar Jena & Disha Trivedi & Harshita Chaudhary & Tapasa Kumar Sahoo & Sriram Seshadri

Received: 8 October 2012 / Accepted: 17 December 2012 / Published online: 1 February 2013 # Springer Science+Business Media New York 2013

Abstract The increase of multidrug-resistant pathogens and the restriction on the use antibiotics due to its side effects have drawn attention to the search for possible alternatives. Bacteriocins are small antimicrobial peptides produced by numerous bacteria. Much interest has been focused on bacteriocins because they exhibit inhibitory activity against pathogens. Lactic acid bacteria possess the ability to synthesize antimicrobial compounds (like bacteriocin) during their growth. In this study, an antibacterial substance (bacteriocin PJ4) produced by Lactobacillus helveticus PJ4, isolated from rat gut microflora, was identified as bacteriocin. It was effective against wide assay of both Gram-positive and Gram-negative bacteria involved in various diseases, including Escherichia coli, Bacillus subtilis, Pseudomonas aeruginosa, Enterococcus faecalis, and Staphylococcus aureus. The antimicrobial peptide was relatively heatresistant and also active over a wide pH range of 2–10. It has been partially purified to homogeneity using ammonium sulfate precipitation and size exclusion chromatography and checked on reverse-phase high-performance liquid chromatography. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis of bacteriocin PJ4 purified through size exclusion chromatography resolved ~6.5 kDa protein with bacteriocin activity. The peptide is inactivated by proteolytic enzymes, trypsin, and lipase but not when treated with catalase, α-amylase, and pepsin. It showed a bactericidal mode of action against the indicator strains E. coli MTCC443, Lactobacillus casei MTCC1423, and E. faecalis DT48. Such characteristics indicate that this bacteriocin may be a potential candidate for alternative agents to control important pathogens.

P. K. Jena : D. Trivedi : H. Chaudhary : S. Seshadri (*) Institute of Science, Nirma University, Sarkhej-Gandhinagar Highway, Chharodi, Ahmedabad 382481 Gujarat, India e-mail: [email protected] T. K. Sahoo School of Life Sciences, Sambalpur University, Jyotivihar, Burla, 768019 Sambalpur, Odisha, India

Author's personal copy Appl Biochem Biotechnol (2013) 169:2088–2100

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Keywords Lactobacillus helveticus . Bacteriocin . Purification . Characterization . Mode of action . Pathogen

Introduction Infections caused by enteric pathogens are an important cause of morbidity and mortality worldwide and have a major impact on public health [1]. Enteric bacteria comprised of Salmonella species, Shigella species, Proteus species, Klebsiella species, Escherichia coli, Pseudomonas species, Vibrio cholerae, and Staphylococcus aureus which are major etiologic agents of enteric infection [2]. Enteric pathogens are often transmitted by means of food or water (foodborne diseases) and are responsible for acute gastroenteritis; some cause systemic disease that may have chronic complications. The rise in antibiotic-resistant bacteria has generated interest in the scientific community to the prophylactic and therapeutic uses of probiotics and to reconsider them as alternatives to antibiotics [3, 4]. Microbial therapeutics is expanding and commensal beneficial bacteria are being implemented as treatment and prevention strategies for immune disorders and infectious diseases [5]. A group of antimicrobials peptides, called bacteriocins, have been studied because they hold a great potential in controlling antibiotic-resistant pathogens. They often act toward species related to the producer with a very high potency and specificity. The common mechanisms of killing by bacteriocins are destruction of target cells by pore formation and/or inhibition of cell wall synthesis [6]. The bacteriocins are active against numerous foodborne and human pathogens, are produced by “generally regarded as safe” microorganisms—Lactobacilli, and are readily degraded by proteolytic host systems, which make them attractive candidates for biotechnological applications [5]. The intestinal microbiota has been identified as a rich source of potential probiotic bacteria which produce novel antimicrobial and, more specifically, antipathogenic bacteriocins having exceptional potential with respect to beneficially modulating the gastrointestinal microbiota and specifically inhibiting specific gastrointestinal pathogens [7]. Different bacteria produce different types of bacteriocins that potentially reach high concentrations in certain local regions of the gut; these compounds act in a nontargeted manner and their contribution to probiotic functionality has not been investigated as extensively. Lactobacillus spp. produce antimicrobial factors and bacteriocins, including lantibiotics, small heatstable, nonlanthionine containing membrane-active peptides, larger heat-labile proteins, and complex bacteriocins containing one or more chemical moieties [8, 9]. Probiotics are producing diverse antimicrobial agents and may be beneficial for the treatment and prevention of a variety of infectious diseases caused by oral, enteric pathogens and urogenital infections [3, 9]. These strains may show therapeutic alternatives in the multidrug-resistant pathogens [10, 11]. Some bacteriocins are active against certain Gram-negative bacteria, such as E. coli and Salmonella Typhimurium [12]. Bacteriocins are ribosomally synthesized and extracellularly released bioactive peptides or proteins displaying antimicrobial activity against other bacteria. They are generally low-molecular-weight proteins that enter into the target cells by binding to cell surface receptors [13]. Therefore, there is an enormous need to explore and isolate more and more bacteria from new sources capable of producing novel bacteriocins and to characterize them for further applications. Very few bacteriocins has been identified and characterized from the Lactobacillus helveticus species. The bacteriocins lactocin LP27 and helveticin J were produced by L. helveticus LP27 and L. helveticus 481, respectively [14,15].

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Appl Biochem Biotechnol (2013) 169:2088–2100

In the present study, we report the purification and characterization of a bacteriocin produced by L. helveticus PJ4, showing bactericidal activity against the indicator organism, enteric E. coli and other selected bacteria. To the best of our knowledge, this is the first report describing the characterization and partial purification of the bacteriocin component of L. helveticus PJ4 isolated from the gut microflora of male Wistar rat (Rattus norvegicus), an experimental animal.

Materials and Methods Bacterial Strains, Growth Conditions, and Media L. helveticus PJ4 (NCBI Accession No. JQ068823) isolated from feces of male Wistar rats was routinely propagated in de Man, Rogosa, and Sharpe (MRS) medium (HiMedia, Mumbai, India). For bacteriocin production, the strain PJ4 was grown in MRS medium. The indicator organism used in bacteriocin assay, E. coli MTCC433, was propagated in brain–heart infusion (BHI) broth. In addition, several Gram-positive and Gram-negative strains (Table 1), from various sources, were used in the determination of spectrum of activity. All cultures were raised at 30–37 °C in MRS or BHI broth as shown in Table 1. All chemicals were obtained from Sigma-Aldrich (USA) and all media components were purchased from HiMedia (India). Antimicrobial activity was determined by the agar spot test method. Activity was expressed as arbitrary units (AU) per milliliter, with 1 AU defined as the reciprocal of the highest dilution showing a clear zone of inhibition [12]. In Vivo Characterization of L. helveticus PJ4 Strain for Probiotics Characteristics Eight- to 10-week-old male Wistar rats weighing 200–250 g were procured from the Laboratory Animals Centre of Zydus Research Center (ZRC), Ahmedabad. Approval to Table 1 Antagonism of bacteriocin PJ4 produced by L. helveticus PJ4 against various target strains Target strains

Collection

Growth medium Growth temperature (°C) Inhibition (mm)

Escherichia coli

MTCC443

BHI

37

Lactobacillus plantarum

fecal isolates

MRS

37

27±0.19

Lactobacillus rhamnosus

MTCC1048

MRS

37

28±0.60

Lactobacillus brevis

Vaginal isolates MRS

37

29±0.28

Lactobacillus casei

MTCC1423

MRS

37

26±0.41

Pseudomonas aeruginosa MTCC1688

BHI

37

17±0.35

Staphylococcus aureus

MTCC737

BHI

37

25±0.32

Enterococcus faecalis Enterococcus faecium

Vaginal isolates MRS Vaginal isolates MRS

30 30

28±0.18 28±0.30

Klebsiella pneumoniae

MTCC109

BHI

37

21±0.27

Salmonella Typhimurium

MTCC733

BHI

37

17±0.15

Shigella flexneri

MTCC1457

BHI

37

18±0.16

Values are presented as the mean ± standard error of the mean (SEM) (n=3) BHI brain–heart infusion agar, MRS de Man, Rogosa, and Sharpe agar (HiMedia)

27±0.45


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work with Wistar rats was obtained from the Ethics Committee of the Nirma University (ethics reference number: IS/BT/PHD10-11/001) from the Ministry of Environment and Forests, Government of India and Committee for the Purpose of Control and Supervision of Experiments on Animals. The animals were acclimatized at a temperature of 25 ± 2 °C and relative humidity of 50–60 % under 12/12 h light/dark conditions for 1 week before experiments. These rats were divided into two groups (six rats each). One group was fed orally with 1 ml of 0.8 % saline containing approximately 107–108 cells of L. helveticus PJ4 daily for a month. Another group fed with 1 ml of saline alone served as a control. Weights of animals were checked weekly. Fecal samples of treated and untreated rats were enumerated for microbiological population. One gram of feces sample was diluted in 0.8 % saline. Diluents (0.1 ml) were spread plated on MRS agar, yeast and mold agar, and violet red bile agar. Plates were incubated for 48 h at 37 °C, and resultant colonies were counted. Mode of Bacteriocin Action Growth of the Test Microorganisms in Presence of Bacteriocin BHI broth was inoculated with 1 % (v/v) E. coli MTCC443; MRS broth was inoculated with 1 % (v/v) Enterococcus faecalis DT48 and Lactobacillus casei MTCC1423 at the early exponential phase and then incubated at 37 °C. The 10-ml filter-sterilized cell-free supernatant was added to the cultures (90 ml), and changes in optical density (at 600 nm) were recorded every hour for 16 h (Fig. 2). Control cells were treated with the inactive bacteriocin (treated for 20 min at 121 °C). Determination of the Reduction of Viable Cells of Target Microorganisms in the Presence of Bacteriocin Early stationary phase cultures of E. coli MTCC443 (16 h old), E. faecalis DT48 (16 h old), and L. casei MTCC1423 (18 h old) were harvested (10,000 rpm, 5 min, 4 °C), washed twice with sterile saline, and resuspended in 10 ml saline. Equal volumes of the cell suspensions and filter-sterilized (0.20-μm; Axiva) bacteriocin PJ4 were mixed. Viable cell numbers were determined before and after incubation for 1 h at 37 °C by plating onto suitable agar medium. Cell suspensions of E. coli MTCC443, E. faecalis DT48, and L. casei MTCC1423 and with no added bacteriocin served as controls. Molecular Mass Determination of Bacteriocin PJ4 A 24-h-old culture of L. helveticus PJ4 obtained in MRS broth at 37 °C was centrifuged (15 min, 10,000 rpm) and the pH adjusted to 6.0 with 1 N NaOH. To avoid proteolytic degradation of the bacteriocin, cell-free supernatants were treated for 10 min at 80 °C. Ammonium sulfate was slowly added to the cell-free supernatants to 60 % saturation and stirred for 4 h at 4 °C and centrifuged (10,000 rpm, 30 min, 4 °C). The precipitate was resuspended in 10 ml of 25 mM ammonium acetate buffer (pH 6.5) and desalted by dialysis using a 1,000-Da cutoff dialysis membrane (Sigma) against the same buffer. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) was used for further separation, as described by Schägger and Von Jagow [16]. Lowmolecular-weight markers with sizes ranging from 2.5 to 45.0 kDa (Amersham Biosciences, Germany) were used.



Bacteriocin Purification Bacteriocin was purified from a 1,000-ml culture of L. helveticus PJ4, grown in MRS broth as described above. Cells were removed by centrifugation at 14,000 rpm for 10 min at room temperature. Purification of bacteriocin was achieved by using a multistep protocol. Ammonium Sulfate Precipitation Culture supernatant was brought to 90 % saturation with solid ammonium sulfate, and after stirring overnight at 4 °C, the precipitate was collected by centrifugation (10,000 rpm, 10 min, 4 °C). The precipitate was dissolved in 60 ml sodium phosphate buffer (20 mM, pH 6.0), and the bacteriocin suspension was desalted by dialyzing through a 2-kDa cutoff dialysis membrane (Sigma) against the same buffer for 24 h. The dialyzed suspension was centrifuged at 14,000 rpm for 15 min at 4 °C. The supernatant was filtered through a 0.2-μm membrane and checked for antimicrobial activity by agar well diffusion assay by using E. coli MTCC443 as the indicator strain and the same was labeled as fraction I. Size Exclusion Chromatography Fraction I was then loaded on a Superdex 75 (10/300 GL) prepacked Tricon column (GE Healthcare, USA) linked to a high-performance liquid chromatography (HPLC; Agilent, 1100) system equilibrated with sodium phosphate buffer (pH 6.7) with 200 mM NaCl at a flow rate of 0.5 ml/min [17]. The eluted peaks were fractionated at a volume 0.5 ml and were checked for antimicrobial activity described earlier. The active fractions were pooled and named as fraction II. Reverse-Phase High-Performance Liquid Chromatography To check the homogeneity of purified active fraction II eluted from the size exclusion chromatography loaded in ACE-5, C-18-300 reverse-phase (RP) column (250×4.6 mm; ACE Capillary Column, Advance Chromatography Technology, Scotland) using an HPLC system (Agilent, USA). The column was equilibrated with solvent A (HPLC-grade water containing 0.1 % trifluoroacetic acid [TFA]). The elution was performed using linear gradient from solvent A to 100 % acetonitrile in 0.1 % TFA (solvent B) for 60 min. The flow rate (0.3 ml/min) and temperature (60 °C) was maintained and eluted analytes were monitored by an ultraviolet detector at 210 nm. Effect of pH, Temperature, and Enzymes on the Activity of Bacteriocin L. helveticus PJ4 was grown in MRS medium for 20 h at 37 °C. The cells were harvested (10,000 rpm, 15 min, 4 °C) and the cell-free supernatant was incubated for 2 h in the presence of trypsin (Sigma), proteinase K (Sigma), pepsin (Sigma), lipase (Sigma), and αamylase (Sigma) at a final concentration of 1.0 mg/ml. Enzyme activity was terminated by boiling for 5 min, and the residual activity was determined by using the well diffusion method. An untreated sample was used as a control (100 %). The effect of pH on the activity of bacteriocin PJ4 was tested by adjusting cell-free supernatants from pH 2.0 to 10.0 (at increments of two pH units) with sterile 1 M NaOH or 1 M HCl for 1 h at 37 °C. After incubation, pH was neutralized to pH 6.0. Antimicrobial activity was tested by well diffusion method. To determine the effects of temperature, purified antimicrobial samples were incubated independently at 30, 45, 60, 75, and 100 ° C for 1 h. A nonheated sample was used as a control (100 %).


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Results and Discussion The aims of this study was to screen the antimicrobial protein producing L. helveticus PJ4 from fecal microflora of male Wistar rats and to screen their bacteriocins as potential natural antibacterial agents for use against few selected pathogens. Spectrum of Activity Bacteriocin PJ4 presented a broad spectrum of activity, being inhibitory against many enteric bacteria and foodborne pathogens. The antibacterial activity of bacteriocin PJ4 was not only evident against Gram-positive bacteria but also against Gram-negative bacteria (Table 1). Similar results were recorded for the cell-free supernatant and for the semipurified bacteriocin (Fig. 3). The bacteriocin PJ4 showed high activity against E. coli, S. aureus, E. faecalis, and Enterococcus faecium but showed low activity against Pseudomonas aeruginosa, Shigella flexneri, Klebsiella pneumoniae, and Salmonella Typhimurium. Most of the bacteriocins described for L. helveticus are active against a much broader range of genera and species [15]. It is important to outline that the bioactivity against E. coli, Salmonella, and Shigella enteric pathogens is of increasing importance. Activity against Gram-negative bacteria is also a relevant characteristic, detected by several authors in other Lactobacilli. Bacteriocin R1333 produced by Lactobacillus sakei R1333 isolated from smoked salmon and enterocin LR/6 produced by E. faecium LR/6 were also active against Gram-negative bacteria [18, 19]. The activity of bacteriocin PJ4 observed against Gram-negative bacteria (Escherichia, Pseudomonas, and Salmonella) is an unpredicted result. Earlier studies [20] reported that bacteriocins of lactic acid bacteria are inefficient to inhibit Gram-negative bacteria because the outer membrane hinders the site for bacteriocin action. However, a few bacteriocins produced by Lactobacillus plantarum have been reported to be active against Gram-negative bacteria. BacteriocinsST26MS (2.8 kDa) and ST28MS (5.5 kDa) produced by L. plantarum ST26MS and ST28MS, respectively, can inhibit Acinetobacter, Escherichia, and Pseudomonas, and antimicrobial activity against Gram-negative bacteria is also a relevant characteristic that was previously detected by several authors in other microorganisms including bacteriocins produced by other Lactobacilli [21]. In Vivo Probiotic Characterization When male Wistar rats were fed with L. helveticus PJ4 (approximately 108 cells/ml for a month), there was no sign of any illness and they were similar as per control rats in terms of weight gain per week. There was a marked decrease in the count of yeast and mold (3.297± 0.23 to 2.889±0.22) and coliform bacteria (6.365±0.15 to 2.795±0.18) in L. helveticus PJ4treated samples compared to untreated rats. A significantly increased count of Lactobacilli was found in treated rat as compared to untreated rats (Table 2). These results indicate the Table 2 Comparison of microbial flora in treated and untreated group with L. helveticus PJ4 Strain

Culture media

L. helveticus PJ4 treated (Log CFU/g)

L. helveticus PJ4 untreated (Log CFU/g)

Lactobacilli

MRS agar

10.084±0.24

8.352±0.27

Yeast and mold Coliforms

Yeast and mold agar Violet red bile agar

2.889±0.22 2.795±0.18

3.297±0.23 6.365±0.15

Values are presented as the mean ± SEM (n=3)



antimicrobial nature of L. helveticus PJ4 inside the gut of the rat. Hence, it could be administered orally as food supplement. Mode of Action E. coli MTCC443 treated with bacteriocin PJ4 (200 AU/ml) increased from OD600 0.145 to 0.459 over 7 h (Fig. 3). The control E. coli MTCC443 (not treated with bacteriocin PJ4) increased from OD600 0.145 to 1.096 over the same period (Fig. 1a). E. faecalis DT48 treated with bacteriocin PJ4 (350 AU/ml) increased from OD600 0.138 to 0.351 over 7 h (Fig. 1b). The control E. faecalis DT48 (not treated with bacteriocin PJ4) increased from OD600 0.137 to 0.725 over the same period. L. casei MTCC1423 treated with bacteriocin PJ4 (600 AU/ml) increased from OD600 0.068 to 0.227over 7 h (Fig. 3). The control L. casei

Fig. 1 The effect of bacteriocin PJ4 on the growth of a E. coli MTCC443, b E. faecalis DT48, and c L. casei MTCC1423. Growth of test microorganisms in presence of bacteriocin PJ4 (triangles) and in the absence of bacteriocin PJ4 (squares). The arrow indicates the time point at which the bacteriocin was added


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MTCC1423 (not treated with bacteriocin PJ4) increased from OD600 0.067 to 0.356 over the same period (Fig. 1c). When stationary-phase cells of E. faecalis DT48, L. casei MTCC1423, and E. coli MTCC443 (107–108 CFU/ml) were treated with the bacteriocin produced by L. helveticus PJ4, complete death of L. casei MTCC1423 and E. coli resulted after 1 h contact time. No viable cells of L. casei MTCC1423 and E. coli MTCC443 were detected, while low levels (102–103 CFU/ml) of viable cells of E. faecalis DT48 were detected. No significant changes in cell numbers of E. faecalis DT48, L. casei MTCC1423, and E. coli were recorded in the untreated (control) sample. Previously, similar results were obtained by bacteriocins HA-6111-2 and HA-5692-3 produced by Pediococcus acidilactici [22], pediocin-like bacteriocin ST5Ha from E. faecium [23], and bacteriocin from L. acidophilus La-14 [24]. Molecular Mass Determination SDS-PAGE was performed for the crude bacteriocin after ammonium sulfate precipitation (Fig. 2) and further by size exclusion chromatography. Due to larger protein mixture (lanes 4 and 5 of Fig. 2b), the ammonium sulfate precipitate bacteriocin was further purified by size exclusion chromatography. According to tricine–SDS-PAGE, bacteriocin PJ4 was estimated to be in the size range of around 6.5 kDa (Fig. 2a). This is within the size range of most bacteriocins reported for the genus Lactobacillus [11]. Bacteriocin Purification Bacteriocin PJ4 produced by L. helveticus PJ4 was purified from cell-free supernatant to homogeneity. By ammonium sulfate precipitation, Tricon column (10/300 GL) Superdex 75 prep grade column gel filtration chromatography, and RP-HPLC (ACE-5, C-18-300, 250× 4.6 mm; ACE Capillary Column) chromatography, the purity of bacteriocin PJ4 was tested.

Fig. 2 SDS-PAGE of bacteriocin PJ4. a Lane 1 molecular mass marker, lane 2 active fractions from size exclusion chromatography of bacteriocin PJ4 stained with Coomassie Blue R250; b lane 3 molecular marker, lanes 4 and 5 SDS-PAGE of silver-stained protein mixture of ammonium sulfate precipitates



Fig. 3 Antimicrobial activity of protein during purification steps: A activity of fraction II of size exclusion chromatography, B activity of fraction1of ammonium sulfate precipitation, C bacteriocin-like peptide with proteinase K, D bacteriocin-like peptide with catalase and proteinase K

Cell-free supernatants from 24-h cultures in MRS broth at 37 °C were used for bacteriocin purification. The activity against E. coli MTCC443 presented by the proteins precipitated with ammonium sulfate and reconstituted in ammonium acetate buffer was similar to that presented by the fractions after size exclusion chromatography on Superdex 75 column. The antimicrobial activity of fraction I and fraction II is shown in Fig. 3. The HPLC analysis of crude bacteriocin after ammonium sulfate precipitation is shown in Fig. 4b. After size exclusion chromatography analysis (Fig. 4b), the active fraction II was reinjected in RPHPLC. A partial purified active fraction was obtained at a retention time of 35 min (Fig. 4c). Similar purification protocol was used by other researchers for purification of bacteriocins [12, 19, 23, 25]. Effect of pH, Temperature, and Enzymes on the Activity of Bacteriocin The antimicrobial protein purified from L. helveticus PJ4 (bacteriocin PJ4) was tested for its sensitivity to pH, temperature, and enzymes; the results are summarized in Table 3. Bacteriocin PJ4 remained stable after incubation for 1 h at pH values from 2.0 to 6.0, but its activity was reduced during pH 8.0–10.0 and highest activity shows at pH 6.0. These results demonstrated that the bacteriocin was resistant to acidic conditions. Bacteriocins were similar with regard to their sensitivity to inactivation by temperature. Like most of the known bacteriocins, they were mainly heat-tolerant at pH 6.5 [26]. The bacteriocin PJ4 showed greater thermal stability. It retained 95.84 % of its initial activity after 1 h incubation at 60 °C, whereas it retained only 69.85 % activity at 100 °C. Only bacteriocins ET30 and ET31 were moderately heat-stable at 60 °C and 100 °C, thus resembling nisin produced by L. lactis WNC20, which was inactivated after 15 min at 121 °C at pH 7.0 [27], or pediocin PA-1, showing about 40 % activity lost after 15 min of heating at 121 °C in the pH range of 2.5–9.0 [28].


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Fig. 4

HPLC chromatogram of primary separation after ammonium sulfate precipitation (a), reinjection in size exclusion chromatography (b), and the active fraction again injected in RP-HPLC for protein purification peak (c) (retention time, 35.63)

The antibacterial activity of bacteriocin PJ4 was completely abolished after treatment with proteolytic enzymes (proteinase K), whereas α-amylase and pepsin had no effect. Treatment of partial purified bacteriocin with catalase did not result in any changes of antibacterial activity (Table 3), indicating that hydrogen peroxide was not responsible for inhibition. These results indicate that bacteriocin PJ4 is a kind of peptide that does not contain lipid or carbohydrate groups. Again, it exhibited high thermal and pH stability. In this case, the secreted organic acids can be ruled out since the pH of the growth medium was always in the neutral range (6.5–7.0). These properties are similar to that of a bacteriocin produced by Lactobacillus sake C2 [29].

Conclusion The antimicrobial activity of lactic acid bacteria may be due to a number of factors, including decreased pH levels, competition for substrates, and production of substances with a bactericidal or bacteriostatic action, including bacteriocins. The antimicrobial components secreted by probiotic strains may help to avoid pathogen colonization of mammalian intestine. Again, these antimicrobial components may find applications as food preservatives and in clinical studies. Table 3 Effects of pH, temperature, and enzymes on the activity of bacteriocin PJ4

Treatment

Residual antimicrobial activity (%) E. coli MTCC443

pH 2

71.23±0.16

4

83.25±0.43

6

91.76±0.23

8

54.17±0.42

10

32.04±0.24

Temperature 30

100

45

100

60

95.84±0.38

75

83.58±0.29

100

69.85±0.37

Enzyme

Values are presented as the mean ± SEM (n=3)

α-Amylase

100

Pepsin Protease K

83.64±0.43 0

Trypsin

0

Lipase

0

Catalase

95±0.38


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This is the first report characterizing the bacteriocin component of L. helveticus PJ4 isolated from the gut microflora of male Wistar rat (R. norvegicus), an experimental animal. The bacteriocin was most effective in inhibiting the growth of various pathogenic strains like E. coli, E. faecalis, S. aureus, E. faecium, K. pneumoniae, and P. aeruginosa. The partially purified bacteriocin was not only biodegradable, but also stable in a wide range of pH values (2.0–10.0), heat-resistant (30–100 °C), and has a size about 6.5 kDa. The bacteriocin PJ4 may have potential application in the prevention and treatment of a few enteric diseases in gastrointestinal tract, which is a major problem in gastrointestinal disorders. However, this needs to be confirmed in future in vivo experiments. In this study, we report the partial characterizations of the antimicrobial compound bacteriocin PJ4; however, the identification and chemical characterizations of these compounds must be carried out to elucidate their complete structure. Acknowledgments The authors are thankful to the Nirma Education and Research Foundation (NERF), Ahmedabad for providing the infrastructure and financial support.

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