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Isolation and biochemical characterisation of a bacteriocin-like substance produced by Bacillus amyloliquefaciens An6
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JGAR-170; No. of Pages 7 Journal of Global Antimicrobial Resistance xxx (2015) xxx–xxx
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Isolation and biochemical characterisation of a bacteriocin-like substance produced by Bacillus amyloliquefaciens An6 Hanen Ben Ayed *, Hana Maalej, Noomen Hmidet, Moncef Nasri Laboratoire de Ge´nie Enzymatique et de Microbiologie, Ecole Nationale d’Inge´nieurs de Sfax, Universite´ de Sfax, B.P. 1173, 3038 Sfax, Tunisia
A R T I C L E I N F O
Article history: Received 4 February 2015 Received in revised form 16 June 2015 Accepted 2 July 2015 Keywords: Bacteriocin Bacillus amyloliquefaciens Biochemical characterisation Antimicrobial activity
A B S T R A C T
This study focuses on the isolation and characterisation of a peptide with bacteriocin-like properties from Bacillus amyloliquefaciens An6. Incubation conditions were optimised, and the effects of the incubation period and of carbon and nitrogen sources were investigated. The produced bacteriocin was partially purified with ammonium sulphate precipitation, dialysis and ultrafiltration and was then biochemically characterised. Maximum bacteriocin production was achieved after 48 h of incubation in a culture medium containing 20 g/L starch and 10 g/L yeast extract, with an initial pH 8.0 at 30 8C under continuous agitation at 200 rpm. The bacteriocin was sequentially purified and its molecular weight was determined to be 11 kDa by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE). The bacteriocin was relatively heat-resistant and was not sensitive to acid and alkaline conditions (pH 4.0–10.0). Its inhibitory activity was sensitive to proteinase K but was resistant to the proteolytic action of alcalase, trypsin, chymotrypsin and pepsin. In conclusion, bacteriocin An6, owing its wide spectrum of activity as well as its high tolerance to acidic and alkaline pH values, temperature and proteases shows great potential for use as a food biopreservative. ß 2015 Published by Elsevier Ltd on behalf of International Society for Chemotherapy of Infection and Cancer.
1. Introduction Bacteriocins are bacterial ribosomally synthesised antimicrobial peptides that are lethal to bacteria other than the producing strain [1]. Owing to their potential use as natural preservatives, bacteriocins produced by lactic acid bacteria have been the subject of intensive investigation in recent years [2]. In contrast, bacteriocins from the genus Bacillus have attracted little attention even though some Bacillus spp., such as Bacillus subtilis and Bacillus licheniformis, are ‘generally recognised as safe’ bacteria [3]. Bacillus is an interesting genus to search for inhibitory substances [4], and Bacillus amyloliquefaciens is one of the major producers of these substances, including several bacteriocins. Unlike bacteriocins produced by lactic acid bacteria, which have a narrow antimicrobial spectrum [5], bacteriocins from Bacillus exhibit distinct diversity in their inhibitory activities [6]. We have screened a number of Bacillus strains isolated from various sources for the production of inhibitory substances against domestic animal pathogens. The selected isolate B.
* Corresponding author. Tel.: +216 96 501 698; fax: +216 74 275 595. E-mail address: [email protected] (H.B. Ayed).
amyloliquefaciens An6 exhibits a potential antimicrobial effect against some important domestic animal pathogens, including Gram-positive and Gram-negative bacteria. In the present study, we report the production, inhibitory spectrum and properties of this antimicrobial compound. 2. Materials and methods 2.1. Antagonistic strain In a previous work, during a screening programme on proteaseproducing strains that have potential industrial applications, B. amyloliquefaciens An6 was isolated from a soil sample collected from an industrial complex/plant producing detergent. It was identified according to the methods described in Bergey’s manual of determinative bacteriology [7] and on the basis of 16S rDNA sequence analysis. It was assigned the GenBank accession no. FJ517583. This strain was found to produce multiple proteases, and fibrinolytic BAF1 enzyme was partially purified and characterised [8]. This strain was also found to produce high amounts of biosurfactants (based on their ability to reduce surface tension from 71 mN/m to 30 mN/m). In the present study, An6 strain was also tested for its ability to produce bacteriocin.
http://dx.doi.org/10.1016/j.jgar.2015.07.001 2213-7165/ß 2015 Published by Elsevier Ltd on behalf of International Society for Chemotherapy of Infection and Cancer.
Please cite this article in press as: Ayed HB, et al. Isolation and biochemical characterisation of a bacteriocin-like substance produced by Bacillus amyloliquefaciens An6. J Global Antimicrob Resist (2015), http://dx.doi.org/10.1016/j.jgar.2015.07.001
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2.2. Antimicrobial activity assay 2.2.1. Agar diffusion method Antibacterial activity was tested against Gram-positive and Gram-negative bacterial strains, including Staphylococcus aureus ATCC 25923, Bacillus cereus ATCC 11778, Enterococcus faecalis ATCC 29212, Micrococcus luteus ATCC 4698, Pseudomonas aeruginosa ATCC 27853, Salmonella typhimurium ATCC 19430, Klebsiella pneumoniae ATCC 13883 and Escherichia coli ATCC 25922. Antifungal activity was tested against Aspergillus niger I1, Mucor rouxii DSM 1191 and Botrytis cinerea. Antimicrobial activity was initially assessed by the agar well diffusion method described by Millette et al. [9]. Culture suspensions of the tested microorganisms were prepared and were serially diluted to 106 CFU/mL for bacterial strains and to 5 104 spores/mL for fungal strains. Luria–Bertani (LB) agar (Sigma Chemical Co., St Louis, MO) and Sabouraud dextrose agar (Sigma Chemical Co.) were used, respectively, for bacterial and fungal strains. Briefly, 100 mL of each suspension was spread over the surface of the LB/Sabouraud plate and was allowed to dry. Wells (7 mm depth, 6 mm diameter) were then cut in the agar and a 60 mL sample (2 mg/L) of the crude bacteriocin-like substance was delivered into them. The plates were then incubated for 24 h at 37 8C for bacteria and for 72 h at 30 8C for fungal strains. The diameter of the inhibition zone was measured and the results are reported in millimetres. The experiment was conducted in triplicate. 2.2.2. MTT test and determination of the minimum inhibitory concentration (MIC) The MIC of bacteriocin An6 against various micro-organisms was determined by a broth microdilution assay. The inoculum of each bacterium was prepared in LB medium and suspensions were adjusted to 106 CFU/mL of bacteria cells estimated by absorbance at 600 nm. Serial dilutions of bacteriocin ranging from 5 mg/mL to 156 mg/mL were prepared in a 96-well plate (MidSci, St Louis, MO). Each well of the sterile microplate included 100 mL of the diluted bacteriocin, 100 mL of the LB growth medium and 10 mL of inoculum. The bacitracin zinc (Bio Basic, Markham, Ontario, Canada) and LB medium were used as positive and negative controls, respectively. Following overnight incubation at 37 8C, growth of the tested micro-organism was monitored at 600 nm by a microtitre plate enzyme-linked immunosorbent assay (ELISA). The MIC was defined as the lowest concentration of sample required for complete inhibition of bacterial growth. 2.3. Bacteriocin production under different conditions Production of antimicrobial activity was first determined in four different media at 30 8C for 48 h: M1 (LB); M2 (yeast extract 10 g/L, glucose 20 g/L); M3 (yeast extract 10 g/L, peptone 10 g/L, casein 20 g/L); and M4 (glucose 20 g/L; (NH4)2SO4 2.3 g/L; K2HPO4 1 g/L; MgSO4 0.5 g/L; KCl 0.5 g/L; glutamic acid 2 g/L, CuSO4 1.6 mg/L; Fe2(SO4)3 1.2 mg/L; MnSO4 0.4 mg/L). At the end of each incubation period, antimicrobial activity was detected by the agar disk diffusion assay. 2.3.1. Optimisation of the culture medium and growth conditions Various carbon sources such as lactose, galactose, sucrose, inulin, starch, maltose and glucose were evaluated for their effect on bacteriocin production by B. amyloliquefaciens An6 strain at a concentration of 2%. The best carbon source was further optimised in the range of 2–30 g/L. The basal medium contained starch as carbon source supplemented with 1% of different organic (casein, yeast extract, soya peptone and pastone) and inorganic nitrogen sources (ammonium sulphate, ammonium chloride and sodium nitrate). The best nitrogen source was further optimised in the
range of 2–20 g/L. The period of incubation (0–72 h), temperature (30 8C and 37 8C) and pH (range 4.0–10.0) were examined for their effect on antagonistic activity production by the An6 strain. 2.3.2. Kinetics of bacteriocin production Flasks containing 25 mL of nutrient broth were inoculated with B. amyloliquefaciens An6 cells from glycerol stock and were incubated at 37 8C in an incubator shaker for 18 h. This was used as a pre-culture inoculum. The kinetics of bacteriocin production was conducted at 30 8C for 72 h in optimised medium (starch 20 g/L and yeast extract 10 g/L). Growth was followed by measuring the optical density at 600 nm (OD600). Samples were withdrawn at desired time intervals by centrifugation at 10 000 g for 15 min and the supernatants were tested for bacteriocin activity against S. aureus. Antagonistic activity was expressed in terms of arbitrary units per millilitre (AU/mL). One arbitrary unit (AU) against an individual indicator strain was defined as the reciprocal of the highest dilution that still produced a minimum detectable zone of inhibition and expressed as AU/mL. 2.4. Partial purification of the bioactive substance Strain An6 was grown in 500 mL of medium containing starch 20 g/L and yeast extract 10 g/L at 30 8C in a rotary shaker at 200 cycles per min for 48 h. Cells were removed by centrifugation at 10 000 g for 15 min. The supernatant was precipitated with ammonium sulphate at 80% saturation under chilled conditions for 18–24 h. The precipitated proteins collected by centrifugation (10 000 g, 30 min), was suspended in 5 mL of 20 mM phosphate buffer solution (pH 6.8) and was dialysed using a 1 kDa cut-off membrane (Sigma Chemical Co.) against the same buffer at 4 8C overnight. The dialysate was then applied to a stirred ultrafiltration cell (Millipore 8400; Merck Millipore, Darmstadt, Germany) using a 10 kDa cut-off membrane (PBGC membrane; Merck Millipore). The fraction with antimicrobial activity obtained was designated as the crude bacteriocin-like substance (CBLS). 2.5. Direct detection of the antimicrobial activity on Tricine–SDSPAGE gels Briefly, 20 mL of the CBLS and low-molecular-mass standards ranging from 3 to 45 kDa were subjected to Tricine–sodium dodecyl sulphate–polyacrylamide gel electrophoresis (Tricine– SDS-PAGE) carried out on 15% polyacrylamide gels using Tricine as trailing ion [10]. Following electrophoresis conducted at 100 V for 90 min, the gel was cut vertically. The first part, containing the sample and protein standards, was stained with staining solution (0.25% Coomassie brilliant blue R-250, 25% isopropanol, 8% acetic acid) to determine the molecular weights of separated protein bands. The other part of the gel was assayed for direct detection of inhibitory activity according to the method described by BarbozaCorona et al. [11] with slight modification. Briefly, this part was washed with phosphate buffer for 3 h. The gel was aseptically placed in a sterile Petri dish with 5 mL of soft brain–heart infusion medium (0.75% agar, w/v) containing ca. 0.1 mL of overnight culture of S. aureus. The Petri dish was incubated at 37 8C for 24 h and was observed for the presence of an inhibition zone. 2.6. Effects of enzymes, heat and pH on antimicrobial activity CBLS was assessed for its sensitivity to proteases and other enzymes. Enzymes (obtained from Sigma Chemical Co.) and their respective buffers were: trypsin, chymotrypsin and alcalase (0.05 M Tris hydrochloride, pH 8.0); proteinase K (1 N NaOH, pH 6.5); pepsin (0.05 M glycine HCl, pH 2.0); and catalase as non-
Please cite this article in press as: Ayed HB, et al. Isolation and biochemical characterisation of a bacteriocin-like substance produced by Bacillus amyloliquefaciens An6. J Global Antimicrob Resist (2015), http://dx.doi.org/10.1016/j.jgar.2015.07.001
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proteolytic enzyme. Samples of CBLS were incubated with different enzymes at a final concentration of 1 mg/mL at 37 8C for 2 h. The antibacterial activity of these different preparations was then determined. Untreated sample, buffer alone and enzyme solutions served as controls. To analyse thermal stability, aliquots of CBLS were incubated at different temperatures in the range of 70–100 8C. After cooling to room temperature, the residual activities were determined. The non-heated CBLS was considered as a control. The effect of pH on CBLS activity was examined by assaying antimicrobial activity using S. aureus following incubation for 1 h at 4 8C in the pH range of 4.0–10.0. Samples were neutralised to pH 7.0 before measurement of the antimicrobial activity. The following buffer systems were used: 100 mM glycine–HCl buffer, pH 2.0–4.0; 100 mM acetate buffer, pH 4.0–6.0; 100 mM Tris–HCl buffer, pH 7.0–8.0; and 100 mM glycine–NaOH buffer, pH 9.0–11.0. 2.7. Cell lysis Cell lysis was studied using the method of De Kwaadsteniet et al. [12]. Briefly, 20 mL of bacteriocin-containing cell-free supernatant was added to a 100 mL culture of S. aureus at the onset of growth (time zero) and again after 2 h of growth. Growth was followed by measuring the OD600 at appropriate intervals. 3. Results and discussion There are many species of the genus Bacillus that can produce a wide variety of antibiotics, including bacitracin, polymyxin, colistin, etc. Bacillus antibiotics are generally produced in the early stages of the sporulation process [13]. Production of antibiotics has been known to be affected by medium components and culture conditions such as carbon source, nitrogen source, agitation, pH and temperature. Deviation from optimal initial pH and temperature for antibiotic production severely affects the yield of production. The agitation rate and medium capacity affect aeration and mixing of the nutrients in the fermentation medium. 3.1. Effect of culture conditions and medium composition on bacteriocin production B. amyloliquefaciens An6 was incubated in several media at 30 8C in a rotary shaker at 200 rpm. Maximum antimicrobial activity against S. aureus was achieved at the middle of the exponential phase by cultivation in M2 medium containing yeast extract 10 g/L and glucose 20 g/L (Table 1). A similar growth profile was observed at 37 8C. However, the maximum activity obtained at 30 8C was greater than that at 37 8C (data not shown). Growth temperature and bacteriocin production are often correlated, as observed for lactocin A [14], enterocin 1146 [15] and nisin Z [16]. The effects of different substrates used as nitrogen and carbon sources on antibiotic production by B. amyloliquefaciens An6 strain were investigated to obtain a suitable medium for maximum production. Lactose, galactose, sucrose, inulin, starch, maltose and glucose were used as carbon sources at a concentration of 20 g/L in Table 1 Production of bacteriocin-like substance by Bacillus amyloliquefaciens An6 in different media. Medium
OD600
IZD (mm)
M1 M2 M3 M4
3.6 4.9 4.17 4.35
12 0.2 23 0.2 15 0.1 12 0.2
OD600, optical density at 600 nm; IZD, inhibition zone diameter. Bacteriocin activity was tested against Staphylococcus aureus.
3
Table 2 Effect of different carbon sources on the production of antimicrobial substance by Bacillus amyloliquefaciens An6. Carbon source
OD600
Protein (mg/mL)
IZD (mm)
Glucose Galactose Lactose Starch Maltose Inulin Sucrose
2.4 2.5 3 4.9 3.1 3.37 2.45
245 4.5 255 5.89 242 5.75 236 8.01 343 6.21 435 5.12 160 4.28
21 0.2 23 0.1 25 0.2 26 0.2 20 0.1 23 0.2 16 0.2
OD600, optical density at 600 nm; IZD, inhibition zone diameter. Cultivation was performed for 48 h at 30 8C in medium consisting of carbon source (20 g/L) and yeast extract (10 g/L).
the presence of 10 g/L yeast extract. Among the carbon sources screened, starch and lactose were found to be the best substrates for antibiotic production. The lowest antimicrobial activity was obtained with sucrose (Table 2). The effect of different concentrations of starch on the antibacterial activity production showed that the maximum was achieved with a concentration of 20 g/L (Table 3). It has been reported by Arquelles-Arias et al. [17] that antibacterial peptide production by B. licheniformis AnBa9 was induced by lactose; however, the level of antibacterial peptide production and its specific activity were gradually decreased by increasing the concentration of lactose. Yu et al. [18] also showed that starch was found to be the most efficient carbon for antifungal antibiotic production by Streptomyces rimosus MY02. The effects of adding organic (yeast extract, casein, soya peptone, pastone) and inorganic (ammonium sulphate, ammonium chloride and sodium nitrate) nitrogen sources at a concentration of 10 g/L were examined in medium containing 20 g/L starch. According to the results shown in Table 4, higher antibiotic production by B. amyloliquefaciens An6 was obtained using yeast extract as nitrogen source. Organic nitrogen sources exerted a favourable effect on antibiotic production compared with inorganic nitrogen sources. These results corroborate well with Table 3 Effect of starch concentration on the production of antimicrobial substance by Bacillus amyloliquefaciens An6. Starch (g/L)
OD600
Protein (mg/mL)
IZD (mm)
0 2 5 10 15 20 25 30
2.40 2.5 3.13 3.71 3.98 5.08 5.23 5.97
118 8.01 186 6.11 221 8.23 245 6.05 289 5.76 236 8.01 364 7.31 310 5.45
17 0.1 17 0.1 18 0.1 18 0.1 22 0.2 26 0.2 24 0.1 21 0.1
OD600, optical density at 600 nm; IZD, inhibition zone diameter. Cultivation was performed for 48 h at 30 8C in medium consisting of different starch concentrations and yeast extract (10 g/L).
Table 4 Effect of different nitrogen sources supplemented to the starch on the production of antimicrobial substance by Bacillus amyloliquefaciens An6. Nitrogen source
OD600
Protein (mg/mL)
IZD (mm)
Yeast extract Pastone Soya peptone NH4Cl NaNO3 (NH4)2SO4 Casein
4.77 3.4 6.73 1.2 4.07 1.28 2.42
236 8.02 200 4.23 221 5.2 5.22 8.12 22.87 7.23 28 10.2 230 8.67
26 0.2 18 0.1 21 0.1 N/D 17 0.1 13 0.2 17 0.2
OD600, optical density at 600 nm; IZD, inhibition zone diameter; N/D, not detected. Cultivation was performed for 48 h at 30 8C in medium consisting of starch (20 g/L) and different nitrogen sources (10 g/L).
Please cite this article in press as: Ayed HB, et al. Isolation and biochemical characterisation of a bacteriocin-like substance produced by Bacillus amyloliquefaciens An6. J Global Antimicrob Resist (2015), http://dx.doi.org/10.1016/j.jgar.2015.07.001
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Table 5 Effect of yeast extract concentration on the production of antimicrobial substance by Bacillus amyloliquefaciens An6. OD600
Protein (mg/mL)
IZD (mm)
0 2 5 10 15 20
1.89 3.47 4.14 5.01 6.47 6.81
48 7.2 136 8.21 147 7.43 226 6.23 317 4.12 320 5.82
14 0.2 18 0.1 20 0.0 26 0.2 23 0.1 22 0.1
OD600, optical density at 600 nm; IZD, inhibition zone diameter. Cultivation was performed for 48 h at 30 8C in medium consisting of starch (20 g/L) different yeast extract concentrations.
IZD (mm)
Yeast extract (g/L)
20 15 10 5 0
4
5
6
7
8
9
10
pH Fig. 1. Effect of initial pH on the production of bacteriocin-like substance by Bacillus amyloliquefaciens An6. Bacteriocin activity was tested against Staphylococcus aureus. IZD, inhibition zone diameter.
previous studies showing that organic nitrogen sources were preferred for compound antibiotic production [17]. The effects of yeast extract concentration on antibiotic production were examined in medium containing starch (20 g/L). As reported in Table 5, antimicrobial activity reached a maximum at 10 g/L yeast extract. This could be due to the availability of larger quantity of free amino acids, short peptides and more growth factors from yeast extract that induce antibiotic compound production. Changes in initial pH affect many cellular processes such as regulation of the biosynthesis of primary and secondary metabolites [19]. To evaluate the effect of initial pH on bacteriocin production, An6 was cultivated in M2 medium at 30 8C at pH values between 4.0 and 10.0 and bacteriocin activity was then measured by the agar disk diffusion assay against S. aureus. Maximum activity (50 U/mL) was detected at pH 7.0, 8.0 and 9.0 (Fig. 1). However, the cell-free extract showed increased inhibition (10 U/mL) after 48 h of incubation at pH 4.0 and 5.0. It has previously been reported by Be´rdy [20] that a pH of 7.8–8.0 gave maximum production of bacitracin. Iglewski and Gerhardt [21] have isolated a strain of B. subtilis with activity against Proteus vulgaris within the pH range 5.7–6.8. 3.2. Inhibitory spectrum The antibacterial activity of the produced CBLS was qualitatively and quantitatively assessed by the presence or absence of inhibition zones as well as determination of zone diameters and MICs compared with that of bacitracin, an antibiotic substance produced by B. licheniformis.
Unlike some bacteriocins from Bacillus spp. that have a relatively narrow inhibitory spectrum [22,23], CBLS showed important antimicrobial activity against micro-organisms with multidrug-resistant profiles (Table 6). Activity against Gramnegative bacteria was lower compared with Gram-positive bacteria. Indeed, the inhibition zones were in the range of 14– 17 mm and 15–21 mm for Gram-negative and Gram-positive bacteria, respectively. B. cereus, S. aureus and S. typhimurium were the most sensitive with halo diameters of 19, 21 and 17 mm, respectively. Furthermore, CBLS exhibited strong inhibitory activity against M. rouxii. At the same concentrations, the positive control bacitracin was found to have the strongest antibacterial activity against Gram-positive bacteria and the opposite trend was observed in the case of Gram-negative bacteria. These results were confirmed by the MICs (Table 6). In fact, the MICs are scientific and significant factors for evaluating the potential of this antimicrobial substance. This method could also compare naturally obtained antimicrobials with commercial antibiotics. The MICs of the produced CBLS ranged between 0.625 mg/mL and 5 mg/mL. Gram-positive bacteria were the most sensitive, with MICs of 1.25, 0.625 and 1.25 mg/mL for S. aureus, B. cereus and M. luteus, respectively. Compared with the positive control, CBLS was more effective against Gram-negative bacteria, especially against E. coli and S. typhimurium, with MICs of 1.25 mg/mL and 2.5 mg/mL, respectively. In the search for antibiotics produced by Bacillus spp., especially B. cereus, B. subtilis and B. licheniformis,
Table 6 Antimicrobial activity spectrum of the inhibitory substance. Indicator strain
Gram-negative bacteria Escherichia coli ATCC 25922 Pseudomonas aeruginosa ATCC 27853 Salmonella typhimurium ATCC 19430 Klebsiella pneumoniae ATCC 13883 Gram-positive bacteria Micrococcus luteus ATCC 4698 Staphylococcus aureus ATCC 25923 Bacillus cereus ATCC 11778 Enterococcus faecalis ATCC 29212 Fungi Aspergillus niger I1 Botrytis cinerea Mucor rouxii DSM 1191
An6 bacteriocin
Bacitracin
IZD (mm)
MIC (mg/mL)
IZD (mm)
MIC (mg/mL)
14 1.0
1.25
7 1.0
<5
17 2.0
2.5 9 1.0
<5
15 2.0 21 1.0 19 1.0
1.25 1.25 0.625 5
19 1.0 25 2.0 22 1.0 19 2.0
0.325 0.156 0.156 0.625
+
IZD, inhibition zone diameter; MIC, minimum inhibitory concentration. , no inhibition. Determinations were performed in triplicate and the data correspond to the mean standard deviation.
Please cite this article in press as: Ayed HB, et al. Isolation and biochemical characterisation of a bacteriocin-like substance produced by Bacillus amyloliquefaciens An6. J Global Antimicrob Resist (2015), http://dx.doi.org/10.1016/j.jgar.2015.07.001
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bacteriocin produced by Enterococcus mundtii, which inhibits the growth of Lactobacillus sakei [12].
Activity (U/ml)
2,4
OD 600 (nm)
50
2
40
1,6
30
1,2
20
0,8
OD (600 nm)
Activity (U/ml)
60
0,4
10
0
0 0
20
40 Time (h)
60
80
Fig. 2. Kinetics of bacteriocin-like substance production. OD600, optical density at 600 nm.
several antimicrobial compounds have been described [24]. Marahiel et al. [25] isolated a B. subtilis strain C126 from sugar cane fermentation that produces the polypeptide antibiotic bacitracin, which inhibited the growth of Micrococcus flavus. B. licheniformis strain 189 isolated from a hot spring environment in the Azores, Portugal, was found to strongly inhibit growth of Grampositive bacteria by producing peptide antibiotic [26]. 3.3. Pattern of antibiotic production by B. amyloliquefaciens An6 To study the production of antimicrobial compound during B. amyloliquefaciens An6 growth, the inhibitory activity present in cell-free samples taken at different time intervals was measured against the indicator strain S. aureus. Antimicrobial activity was detected at the mid-log growth phase and quickly reached a maximum at the early stationary phase (Fig. 2). After that, the antagonistic activity declined. This decrease may be attributed to adsorption on producer cells or to degradation by specific or nonspecific proteases [14]. The kinetics of this production was similar to those described for bacteriocins from Bacillus thuringiensis subsp. tochigiensis [22]. 3.4. Study of mode of action of antimicrobial compound produced by B. amyloliquefaciens strain An6 The mode of action of the CBLS was studied using S. aureus as the target bacteria. The inhibitory activity on the growth of S. aureus is shown in Fig. 3. Addition of CBLS to ca. 106 CFU/mL of indicator strain resulted in growth inhibition. Indeed, the OD600 of the treated culture changed significantly during the experiment. Cell lysis has been recorded when bacteriocin was added to actively growing cells of S. aureus, suggesting that the mode of action is bactericidal. A similar mode of action was reported for the 1
Control Bacteriocin added at 0 h Bacteriocin added after 2 h
OD (600nm)
0,8 0,6
CBLS
0,4
CBLS
0,2 0 0
2
4
6
8
5
10
Time (h) Fig. 3. Study of the mode of action of bacteriocin-like substance An6. OD, optical density; CBLS, crude bacteriocin-like substance.
3.5. Effects of enzymes, heat and pH on antimicrobial activity To determine the biochemical and biophysical properties of the inhibitory substance, samples were tested for sensitivity to enzymes, temperature and pH and the results are summarised in Table 7. Little or no change in the size of the inhibitory zone was observed when the same samples were treated with alcalase, trypsin, chymotrypsin, pepsin or catalase. There was a significant loss of inhibitory activity after treatment with proteinase K and catalase. The antimicrobial activity was sensitive to proteinase K and was unaffected by catalase activity, suggesting that it is proteinaceous in nature. These results suggest that this antimicrobial peptide possibly can survive in the intestinal environment and could be administered with feed. These results were different to bacteriocin-like substance produced by B. amyloliquefaciens isolated from the Brazilian Atlantic forest [27], which is sensitive to the proteolytic action of trypsin, papain, proteinase K and pronase E. The heat sensitivity of the antimicrobial substance was determined by measuring its activity following incubation for 15 min at different temperatures. It was stable at temperatures up to 80 8C but activity decreased at 100 8C and the substance was completely inactivated after incubation for 45 min at 121 8C. Lyophilisation and re-suspension of the sample did not affect the antimicrobial activity, indicating that this bioactive compound is attractive for potential biotechnological application. To evaluate the antimicrobial substance stability at different pH values, the CBLS was incubated at 25 8C at pH values of 4.0–10.0 for 2 h and activity was measured by the agar disk diffusion assay against S. aureus (Table 7). Activity was retained over a pH range of 4.0–10.0. These results were similar to coagulin, a bacteriocin-like inhibitory substance produced by Bacillus coagulans [28]. Bacteriocin An6, owing its wide activity spectrum and its high tolerance to acidic and alkaline pH values, temperature and proteases shows great potential for use as a food biopreservative. Food preservation has become a major issue because foodborne pathogens can cause havoc in preserved/fresh food items at high temperature, room temperature and even at low temperature. However, consumer demand for faster, healthier and ready-to-eat
Table 7 Effect of enzymes, heat and pH on antimicrobial activity. Treatment
Residual activity (%)a
Enzymes Proteinase K Trypsin Chymotrypsin Alcalase Pepsin
45 90 100 100 85
Heat/time 70 8C/15 min 80 8C/15 min 90 8C/15 min 100 8C/15 min 121 8C/45 min
100 100 95 70 –
pH 4 6 8 10
90 100 100 85
a Untreated crude bacteriocin-like substance (CBLS) was considered as 100% and the residual activity was defined as the ratio (treated/untreated CBLS).
Please cite this article in press as: Ayed HB, et al. Isolation and biochemical characterisation of a bacteriocin-like substance produced by Bacillus amyloliquefaciens An6. J Global Antimicrob Resist (2015), http://dx.doi.org/10.1016/j.jgar.2015.07.001
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of the abovementioned results, this antimicrobial substance shows a potential to be used in the medical and food industries. Stability under extreme pH values has an advantage for application in food as a biopreservative. Furthermore, heat resistance is an advantage since bacteriocins may remain active in foods after cooking and give protection against undesirable bacteria. Resistance to protease hydrolysis suggests that this antimicrobial peptide possibly can survive in the intestinal environment and could be administered with feed. Funding This work was funded by the Ministry of Higher Education and Scientific Research, Tunisia. Competing interests None declared. Ethical approval Not required. Fig. 4. Tricine–SDS-PAGE analysis and direct detection of antimicrobial activity. (A) Gel stained with Coomassie blue: lane 1, molecular weight marker; lane 2, crude supernatant. (B) The inhibition zone was shown by overlaying the gel with soft brain–heart infusion medium (0.75% agar, w/v) containing the Staphylococcus aureus indicator strain.
products has strongly demanded the use of more natural preservatives instead of chemical preservatives. Microbiologists became interested in bacteriocin-producing micro-organisms to overcome this problem that fulfil the requirement of food preservation. In this study, we have characterised a bacteriocinlike substance produced by B. amyloliquefaciens An6 strain that has a large inhibitory spectrum. This bacteriocin presents a wide spectrum of activity covering Gram-positive bacteria, Gramnegative bacteria and fungi. Considering the bacteriocin properties, its antimicrobial activity was stable over a wide temperature range from 70 to 100 8C and it was highly active over a wide range of pH from 4.0 to 10.0. Thus, this antimicrobial substance can be incorporated into food products to control microbial growth and to enhance the safety and shelf-life of food products. Heat stability is a very useful characteristic in the application of bacteriocin as a food preservative because many food-processing procedures involve a heating step. 3.6. SDS-PAGE analysis and mode of action of the inhibitory substance The dialysate generated after precipitation with ammonium sulphate was fractionated by ultrafiltration by using 10 kDa molecular weight cut-off membranes. Two fractions were obtained (concentrate and permeate). The antimicrobial activity was recovered in the concentrate, which contains peptides with a molecular weight >10 kDa. This results indicates that the active unit has a molecular mass >10 kDa. Direct detection of the antimicrobial activity was performed by Tricine–SDS-PAGE. As shown in the stained bands of the gel, several proteins were detected in the samples (Fig. 4A). Overlaying the gel with the indicator strain revealed a single protein band with inhibitory activity (Fig. 4B). The band had an apparent molecular mass of ca. 11 kDa. 4. Conclusion Here we report the production and characterisation of a bacteriocin-like substance from B. amyloliquefaciens. On the basis
References [1] Joerger RD. Alternatives to antibiotics: bacteriocins, antimicrobial peptides and bacteriophages. Poult Sci 2003;82:640–7. [2] Eijsink VGH, Skeie M, Middelhoven PH, Brurberg MB, Nes IF. Comparative studies of class IIa bacteriocins of lactic acid bacteria. Appl Environ Microbiol 1998;64:3275–81. [3] Teo AYL, Tan HM. Inhibition of Clostridium perfringens by a novel strain of Bacillus subtilis isolated from the gastrointestinal tracts of healthy chickens. Appl Environ Microbiol 2005;71:4185–90. [4] Bizani D, Dominguez APM, Brandelli A. Purification and partial chemical characterization of the antimicrobial peptide cerein 8A. Lett Appl Microbiol 2005;41:269–73. [5] Jack RW, Tagg JR, Ray B. Bacteriocins of Gram-positive bacteria. Microbiol Rev 1995;59:171–200. [6] Cordovilla P, Valdivia E, Gonzalez-Sequra A, Galvez A, Martinez-Bueno M, Maqueda M. Antagonistic action of the bacterium Bacillus licheniformis M-4 toward the amoeba Naegleria fowleri. J Eukaryot Microbiol 1993;40:323–8. [7] Claus D, Berkeley RCW. Genus Bacillus Cohn 1872. In: Sneath PHA, et al., editors. Bergey’s manual of determinative bacteriology, vol 2. Baltimore, MD: Williams and Wilkins Co.; 1986. p. 1105–39. [8] Agrebi R, Hmidet N, Hajji M, Ktari N, Haddar A, Fakhfakh-Zouari N, et al. Fibrinolytic serine protease isolation from Bacillus amyloliquefaciens An6 grown on Mirabilis jalapa tuber powders. Appl Biochem Biotechnol 2010;162:75–88. [9] Millette M, Dupont C, Archambault D, Lacroix M. Partial characterization of bacteriocins produced by human Lactococcus lactis and Pediococcus acidilactici isolates. J Appl Microbiol 2007;102:274–82. [10] Scha¨gger H. Tricine–SDS-PAGE. Nat Protoc 2006;1:16–22. [11] Barboza-Corona JE, Va´zquez-Acosta H, Bideshi DK, Salcedo-Herna´ndez R. Bacteriocin-like inhibitor substances produced by Mexican strains of Bacillus thuringiensis. Arch Microbiol 2007;187:117–26. [12] De Kwaadsteniet M, Todorov D, Knoetze H, Dicks LMT. Characterization of a 3944 Da bacteriocin, produced by Enterococcus mundtii ST15, with activity against Gram-positive and Gram-negative bacteria. Int J Food Microbiol 2005;41:433–44. [13] Bizani D, Brandelli A. Characterization of a bacteriocin produced by a newly isolated Bacillus sp. strain 8A. J Appl Microbiol 2002;93:512–9. [14] Parente E, Ricciardi A, Addario G. Influence of pH on growth and bacteriocin production by Lactococcus lactis subsp. lactis 140NWC during batch fermentation. Appl Microbiol Biotechnol 1994;41:388–94. [15] Parente E, Ricciardi A. Production, recovery and purification of bacteriocins from lactic acid bacteria. Appl Microbiol Biotechnol 1994;52:628–38. [16] Matsusaki H, Endo N, Sonomoto K, Ishizaki A. Lantibiotic nisin Z fermentative production by Lactococcus lactis IO-1: relationship between production of the lantibiotic and lactate and cell growth. Appl Microbiol Biotechnol 1996;45: 36–40. [17] Arquelles-Arias A, Ongena M, Halimi B, Lara Y, Brans A, Joris B, et al. Bacillus amyloliquefaciens GA1 as a source of potent antibiotics and other secondary metabolites for biocontrol of plant pathogens. Microb Cell Fact 2009;8:63. [18] Yu J, Liu Q, Liu Q, Liu X, Sun Q, Yan J, et al. Effect of liquid culture requirements on antifungal antibiotic production by Streptomyces rimosus MY02. Bioresour Technol 2008;99:2087–91. [19] Sole´ M, Francia A, Rius N, Lore´n JG. The role of pH in the ‘glucose effect’ on prodigiosin production by non-proliferating cells of Serratia marcescens. Lett Appl Microbiol 1997;25:81–4.
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Isolation and biochemical characterisation of a bacteriocin-like substance produced by Bacillus amyloliquefaciens An6 Hanen Ben Ayed *, Hana Maalej, Noomen Hmidet, Moncef Nasri Laboratoire de Ge´nie Enzymatique et de Microbiologie, Ecole Nationale d’Inge´nieurs de Sfax, Universite´ de Sfax, B.P. 1173, 3038 Sfax, Tunisia
A R T I C L E I N F O
Article history: Received 4 February 2015 Received in revised form 16 June 2015 Accepted 2 July 2015 Keywords: Bacteriocin Bacillus amyloliquefaciens Biochemical characterisation Antimicrobial activity
A B S T R A C T
This study focuses on the isolation and characterisation of a peptide with bacteriocin-like properties from Bacillus amyloliquefaciens An6. Incubation conditions were optimised, and the effects of the incubation period and of carbon and nitrogen sources were investigated. The produced bacteriocin was partially purified with ammonium sulphate precipitation, dialysis and ultrafiltration and was then biochemically characterised. Maximum bacteriocin production was achieved after 48 h of incubation in a culture medium containing 20 g/L starch and 10 g/L yeast extract, with an initial pH 8.0 at 30 8C under continuous agitation at 200 rpm. The bacteriocin was sequentially purified and its molecular weight was determined to be 11 kDa by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE). The bacteriocin was relatively heat-resistant and was not sensitive to acid and alkaline conditions (pH 4.0–10.0). Its inhibitory activity was sensitive to proteinase K but was resistant to the proteolytic action of alcalase, trypsin, chymotrypsin and pepsin. In conclusion, bacteriocin An6, owing its wide spectrum of activity as well as its high tolerance to acidic and alkaline pH values, temperature and proteases shows great potential for use as a food biopreservative. ß 2015 Published by Elsevier Ltd on behalf of International Society for Chemotherapy of Infection and Cancer.
1. Introduction Bacteriocins are bacterial ribosomally synthesised antimicrobial peptides that are lethal to bacteria other than the producing strain [1]. Owing to their potential use as natural preservatives, bacteriocins produced by lactic acid bacteria have been the subject of intensive investigation in recent years [2]. In contrast, bacteriocins from the genus Bacillus have attracted little attention even though some Bacillus spp., such as Bacillus subtilis and Bacillus licheniformis, are ‘generally recognised as safe’ bacteria [3]. Bacillus is an interesting genus to search for inhibitory substances [4], and Bacillus amyloliquefaciens is one of the major producers of these substances, including several bacteriocins. Unlike bacteriocins produced by lactic acid bacteria, which have a narrow antimicrobial spectrum [5], bacteriocins from Bacillus exhibit distinct diversity in their inhibitory activities [6]. We have screened a number of Bacillus strains isolated from various sources for the production of inhibitory substances against domestic animal pathogens. The selected isolate B.
* Corresponding author. Tel.: +216 96 501 698; fax: +216 74 275 595. E-mail address: [email protected] (H.B. Ayed).
amyloliquefaciens An6 exhibits a potential antimicrobial effect against some important domestic animal pathogens, including Gram-positive and Gram-negative bacteria. In the present study, we report the production, inhibitory spectrum and properties of this antimicrobial compound. 2. Materials and methods 2.1. Antagonistic strain In a previous work, during a screening programme on proteaseproducing strains that have potential industrial applications, B. amyloliquefaciens An6 was isolated from a soil sample collected from an industrial complex/plant producing detergent. It was identified according to the methods described in Bergey’s manual of determinative bacteriology [7] and on the basis of 16S rDNA sequence analysis. It was assigned the GenBank accession no. FJ517583. This strain was found to produce multiple proteases, and fibrinolytic BAF1 enzyme was partially purified and characterised [8]. This strain was also found to produce high amounts of biosurfactants (based on their ability to reduce surface tension from 71 mN/m to 30 mN/m). In the present study, An6 strain was also tested for its ability to produce bacteriocin.
http://dx.doi.org/10.1016/j.jgar.2015.07.001 2213-7165/ß 2015 Published by Elsevier Ltd on behalf of International Society for Chemotherapy of Infection and Cancer.
Please cite this article in press as: Ayed HB, et al. Isolation and biochemical characterisation of a bacteriocin-like substance produced by Bacillus amyloliquefaciens An6. J Global Antimicrob Resist (2015), http://dx.doi.org/10.1016/j.jgar.2015.07.001
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2.2. Antimicrobial activity assay 2.2.1. Agar diffusion method Antibacterial activity was tested against Gram-positive and Gram-negative bacterial strains, including Staphylococcus aureus ATCC 25923, Bacillus cereus ATCC 11778, Enterococcus faecalis ATCC 29212, Micrococcus luteus ATCC 4698, Pseudomonas aeruginosa ATCC 27853, Salmonella typhimurium ATCC 19430, Klebsiella pneumoniae ATCC 13883 and Escherichia coli ATCC 25922. Antifungal activity was tested against Aspergillus niger I1, Mucor rouxii DSM 1191 and Botrytis cinerea. Antimicrobial activity was initially assessed by the agar well diffusion method described by Millette et al. [9]. Culture suspensions of the tested microorganisms were prepared and were serially diluted to 106 CFU/mL for bacterial strains and to 5 104 spores/mL for fungal strains. Luria–Bertani (LB) agar (Sigma Chemical Co., St Louis, MO) and Sabouraud dextrose agar (Sigma Chemical Co.) were used, respectively, for bacterial and fungal strains. Briefly, 100 mL of each suspension was spread over the surface of the LB/Sabouraud plate and was allowed to dry. Wells (7 mm depth, 6 mm diameter) were then cut in the agar and a 60 mL sample (2 mg/L) of the crude bacteriocin-like substance was delivered into them. The plates were then incubated for 24 h at 37 8C for bacteria and for 72 h at 30 8C for fungal strains. The diameter of the inhibition zone was measured and the results are reported in millimetres. The experiment was conducted in triplicate. 2.2.2. MTT test and determination of the minimum inhibitory concentration (MIC) The MIC of bacteriocin An6 against various micro-organisms was determined by a broth microdilution assay. The inoculum of each bacterium was prepared in LB medium and suspensions were adjusted to 106 CFU/mL of bacteria cells estimated by absorbance at 600 nm. Serial dilutions of bacteriocin ranging from 5 mg/mL to 156 mg/mL were prepared in a 96-well plate (MidSci, St Louis, MO). Each well of the sterile microplate included 100 mL of the diluted bacteriocin, 100 mL of the LB growth medium and 10 mL of inoculum. The bacitracin zinc (Bio Basic, Markham, Ontario, Canada) and LB medium were used as positive and negative controls, respectively. Following overnight incubation at 37 8C, growth of the tested micro-organism was monitored at 600 nm by a microtitre plate enzyme-linked immunosorbent assay (ELISA). The MIC was defined as the lowest concentration of sample required for complete inhibition of bacterial growth. 2.3. Bacteriocin production under different conditions Production of antimicrobial activity was first determined in four different media at 30 8C for 48 h: M1 (LB); M2 (yeast extract 10 g/L, glucose 20 g/L); M3 (yeast extract 10 g/L, peptone 10 g/L, casein 20 g/L); and M4 (glucose 20 g/L; (NH4)2SO4 2.3 g/L; K2HPO4 1 g/L; MgSO4 0.5 g/L; KCl 0.5 g/L; glutamic acid 2 g/L, CuSO4 1.6 mg/L; Fe2(SO4)3 1.2 mg/L; MnSO4 0.4 mg/L). At the end of each incubation period, antimicrobial activity was detected by the agar disk diffusion assay. 2.3.1. Optimisation of the culture medium and growth conditions Various carbon sources such as lactose, galactose, sucrose, inulin, starch, maltose and glucose were evaluated for their effect on bacteriocin production by B. amyloliquefaciens An6 strain at a concentration of 2%. The best carbon source was further optimised in the range of 2–30 g/L. The basal medium contained starch as carbon source supplemented with 1% of different organic (casein, yeast extract, soya peptone and pastone) and inorganic nitrogen sources (ammonium sulphate, ammonium chloride and sodium nitrate). The best nitrogen source was further optimised in the
range of 2–20 g/L. The period of incubation (0–72 h), temperature (30 8C and 37 8C) and pH (range 4.0–10.0) were examined for their effect on antagonistic activity production by the An6 strain. 2.3.2. Kinetics of bacteriocin production Flasks containing 25 mL of nutrient broth were inoculated with B. amyloliquefaciens An6 cells from glycerol stock and were incubated at 37 8C in an incubator shaker for 18 h. This was used as a pre-culture inoculum. The kinetics of bacteriocin production was conducted at 30 8C for 72 h in optimised medium (starch 20 g/L and yeast extract 10 g/L). Growth was followed by measuring the optical density at 600 nm (OD600). Samples were withdrawn at desired time intervals by centrifugation at 10 000 g for 15 min and the supernatants were tested for bacteriocin activity against S. aureus. Antagonistic activity was expressed in terms of arbitrary units per millilitre (AU/mL). One arbitrary unit (AU) against an individual indicator strain was defined as the reciprocal of the highest dilution that still produced a minimum detectable zone of inhibition and expressed as AU/mL. 2.4. Partial purification of the bioactive substance Strain An6 was grown in 500 mL of medium containing starch 20 g/L and yeast extract 10 g/L at 30 8C in a rotary shaker at 200 cycles per min for 48 h. Cells were removed by centrifugation at 10 000 g for 15 min. The supernatant was precipitated with ammonium sulphate at 80% saturation under chilled conditions for 18–24 h. The precipitated proteins collected by centrifugation (10 000 g, 30 min), was suspended in 5 mL of 20 mM phosphate buffer solution (pH 6.8) and was dialysed using a 1 kDa cut-off membrane (Sigma Chemical Co.) against the same buffer at 4 8C overnight. The dialysate was then applied to a stirred ultrafiltration cell (Millipore 8400; Merck Millipore, Darmstadt, Germany) using a 10 kDa cut-off membrane (PBGC membrane; Merck Millipore). The fraction with antimicrobial activity obtained was designated as the crude bacteriocin-like substance (CBLS). 2.5. Direct detection of the antimicrobial activity on Tricine–SDSPAGE gels Briefly, 20 mL of the CBLS and low-molecular-mass standards ranging from 3 to 45 kDa were subjected to Tricine–sodium dodecyl sulphate–polyacrylamide gel electrophoresis (Tricine– SDS-PAGE) carried out on 15% polyacrylamide gels using Tricine as trailing ion [10]. Following electrophoresis conducted at 100 V for 90 min, the gel was cut vertically. The first part, containing the sample and protein standards, was stained with staining solution (0.25% Coomassie brilliant blue R-250, 25% isopropanol, 8% acetic acid) to determine the molecular weights of separated protein bands. The other part of the gel was assayed for direct detection of inhibitory activity according to the method described by BarbozaCorona et al. [11] with slight modification. Briefly, this part was washed with phosphate buffer for 3 h. The gel was aseptically placed in a sterile Petri dish with 5 mL of soft brain–heart infusion medium (0.75% agar, w/v) containing ca. 0.1 mL of overnight culture of S. aureus. The Petri dish was incubated at 37 8C for 24 h and was observed for the presence of an inhibition zone. 2.6. Effects of enzymes, heat and pH on antimicrobial activity CBLS was assessed for its sensitivity to proteases and other enzymes. Enzymes (obtained from Sigma Chemical Co.) and their respective buffers were: trypsin, chymotrypsin and alcalase (0.05 M Tris hydrochloride, pH 8.0); proteinase K (1 N NaOH, pH 6.5); pepsin (0.05 M glycine HCl, pH 2.0); and catalase as non-
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proteolytic enzyme. Samples of CBLS were incubated with different enzymes at a final concentration of 1 mg/mL at 37 8C for 2 h. The antibacterial activity of these different preparations was then determined. Untreated sample, buffer alone and enzyme solutions served as controls. To analyse thermal stability, aliquots of CBLS were incubated at different temperatures in the range of 70–100 8C. After cooling to room temperature, the residual activities were determined. The non-heated CBLS was considered as a control. The effect of pH on CBLS activity was examined by assaying antimicrobial activity using S. aureus following incubation for 1 h at 4 8C in the pH range of 4.0–10.0. Samples were neutralised to pH 7.0 before measurement of the antimicrobial activity. The following buffer systems were used: 100 mM glycine–HCl buffer, pH 2.0–4.0; 100 mM acetate buffer, pH 4.0–6.0; 100 mM Tris–HCl buffer, pH 7.0–8.0; and 100 mM glycine–NaOH buffer, pH 9.0–11.0. 2.7. Cell lysis Cell lysis was studied using the method of De Kwaadsteniet et al. [12]. Briefly, 20 mL of bacteriocin-containing cell-free supernatant was added to a 100 mL culture of S. aureus at the onset of growth (time zero) and again after 2 h of growth. Growth was followed by measuring the OD600 at appropriate intervals. 3. Results and discussion There are many species of the genus Bacillus that can produce a wide variety of antibiotics, including bacitracin, polymyxin, colistin, etc. Bacillus antibiotics are generally produced in the early stages of the sporulation process [13]. Production of antibiotics has been known to be affected by medium components and culture conditions such as carbon source, nitrogen source, agitation, pH and temperature. Deviation from optimal initial pH and temperature for antibiotic production severely affects the yield of production. The agitation rate and medium capacity affect aeration and mixing of the nutrients in the fermentation medium. 3.1. Effect of culture conditions and medium composition on bacteriocin production B. amyloliquefaciens An6 was incubated in several media at 30 8C in a rotary shaker at 200 rpm. Maximum antimicrobial activity against S. aureus was achieved at the middle of the exponential phase by cultivation in M2 medium containing yeast extract 10 g/L and glucose 20 g/L (Table 1). A similar growth profile was observed at 37 8C. However, the maximum activity obtained at 30 8C was greater than that at 37 8C (data not shown). Growth temperature and bacteriocin production are often correlated, as observed for lactocin A [14], enterocin 1146 [15] and nisin Z [16]. The effects of different substrates used as nitrogen and carbon sources on antibiotic production by B. amyloliquefaciens An6 strain were investigated to obtain a suitable medium for maximum production. Lactose, galactose, sucrose, inulin, starch, maltose and glucose were used as carbon sources at a concentration of 20 g/L in Table 1 Production of bacteriocin-like substance by Bacillus amyloliquefaciens An6 in different media. Medium
OD600
IZD (mm)
M1 M2 M3 M4
3.6 4.9 4.17 4.35
12 0.2 23 0.2 15 0.1 12 0.2
OD600, optical density at 600 nm; IZD, inhibition zone diameter. Bacteriocin activity was tested against Staphylococcus aureus.
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Table 2 Effect of different carbon sources on the production of antimicrobial substance by Bacillus amyloliquefaciens An6. Carbon source
OD600
Protein (mg/mL)
IZD (mm)
Glucose Galactose Lactose Starch Maltose Inulin Sucrose
2.4 2.5 3 4.9 3.1 3.37 2.45
245 4.5 255 5.89 242 5.75 236 8.01 343 6.21 435 5.12 160 4.28
21 0.2 23 0.1 25 0.2 26 0.2 20 0.1 23 0.2 16 0.2
OD600, optical density at 600 nm; IZD, inhibition zone diameter. Cultivation was performed for 48 h at 30 8C in medium consisting of carbon source (20 g/L) and yeast extract (10 g/L).
the presence of 10 g/L yeast extract. Among the carbon sources screened, starch and lactose were found to be the best substrates for antibiotic production. The lowest antimicrobial activity was obtained with sucrose (Table 2). The effect of different concentrations of starch on the antibacterial activity production showed that the maximum was achieved with a concentration of 20 g/L (Table 3). It has been reported by Arquelles-Arias et al. [17] that antibacterial peptide production by B. licheniformis AnBa9 was induced by lactose; however, the level of antibacterial peptide production and its specific activity were gradually decreased by increasing the concentration of lactose. Yu et al. [18] also showed that starch was found to be the most efficient carbon for antifungal antibiotic production by Streptomyces rimosus MY02. The effects of adding organic (yeast extract, casein, soya peptone, pastone) and inorganic (ammonium sulphate, ammonium chloride and sodium nitrate) nitrogen sources at a concentration of 10 g/L were examined in medium containing 20 g/L starch. According to the results shown in Table 4, higher antibiotic production by B. amyloliquefaciens An6 was obtained using yeast extract as nitrogen source. Organic nitrogen sources exerted a favourable effect on antibiotic production compared with inorganic nitrogen sources. These results corroborate well with Table 3 Effect of starch concentration on the production of antimicrobial substance by Bacillus amyloliquefaciens An6. Starch (g/L)
OD600
Protein (mg/mL)
IZD (mm)
0 2 5 10 15 20 25 30
2.40 2.5 3.13 3.71 3.98 5.08 5.23 5.97
118 8.01 186 6.11 221 8.23 245 6.05 289 5.76 236 8.01 364 7.31 310 5.45
17 0.1 17 0.1 18 0.1 18 0.1 22 0.2 26 0.2 24 0.1 21 0.1
OD600, optical density at 600 nm; IZD, inhibition zone diameter. Cultivation was performed for 48 h at 30 8C in medium consisting of different starch concentrations and yeast extract (10 g/L).
Table 4 Effect of different nitrogen sources supplemented to the starch on the production of antimicrobial substance by Bacillus amyloliquefaciens An6. Nitrogen source
OD600
Protein (mg/mL)
IZD (mm)
Yeast extract Pastone Soya peptone NH4Cl NaNO3 (NH4)2SO4 Casein
4.77 3.4 6.73 1.2 4.07 1.28 2.42
236 8.02 200 4.23 221 5.2 5.22 8.12 22.87 7.23 28 10.2 230 8.67
26 0.2 18 0.1 21 0.1 N/D 17 0.1 13 0.2 17 0.2
OD600, optical density at 600 nm; IZD, inhibition zone diameter; N/D, not detected. Cultivation was performed for 48 h at 30 8C in medium consisting of starch (20 g/L) and different nitrogen sources (10 g/L).
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Table 5 Effect of yeast extract concentration on the production of antimicrobial substance by Bacillus amyloliquefaciens An6. OD600
Protein (mg/mL)
IZD (mm)
0 2 5 10 15 20
1.89 3.47 4.14 5.01 6.47 6.81
48 7.2 136 8.21 147 7.43 226 6.23 317 4.12 320 5.82
14 0.2 18 0.1 20 0.0 26 0.2 23 0.1 22 0.1
OD600, optical density at 600 nm; IZD, inhibition zone diameter. Cultivation was performed for 48 h at 30 8C in medium consisting of starch (20 g/L) different yeast extract concentrations.
IZD (mm)
Yeast extract (g/L)
20 15 10 5 0
4
5
6
7
8
9
10
pH Fig. 1. Effect of initial pH on the production of bacteriocin-like substance by Bacillus amyloliquefaciens An6. Bacteriocin activity was tested against Staphylococcus aureus. IZD, inhibition zone diameter.
previous studies showing that organic nitrogen sources were preferred for compound antibiotic production [17]. The effects of yeast extract concentration on antibiotic production were examined in medium containing starch (20 g/L). As reported in Table 5, antimicrobial activity reached a maximum at 10 g/L yeast extract. This could be due to the availability of larger quantity of free amino acids, short peptides and more growth factors from yeast extract that induce antibiotic compound production. Changes in initial pH affect many cellular processes such as regulation of the biosynthesis of primary and secondary metabolites [19]. To evaluate the effect of initial pH on bacteriocin production, An6 was cultivated in M2 medium at 30 8C at pH values between 4.0 and 10.0 and bacteriocin activity was then measured by the agar disk diffusion assay against S. aureus. Maximum activity (50 U/mL) was detected at pH 7.0, 8.0 and 9.0 (Fig. 1). However, the cell-free extract showed increased inhibition (10 U/mL) after 48 h of incubation at pH 4.0 and 5.0. It has previously been reported by Be´rdy [20] that a pH of 7.8–8.0 gave maximum production of bacitracin. Iglewski and Gerhardt [21] have isolated a strain of B. subtilis with activity against Proteus vulgaris within the pH range 5.7–6.8. 3.2. Inhibitory spectrum The antibacterial activity of the produced CBLS was qualitatively and quantitatively assessed by the presence or absence of inhibition zones as well as determination of zone diameters and MICs compared with that of bacitracin, an antibiotic substance produced by B. licheniformis.
Unlike some bacteriocins from Bacillus spp. that have a relatively narrow inhibitory spectrum [22,23], CBLS showed important antimicrobial activity against micro-organisms with multidrug-resistant profiles (Table 6). Activity against Gramnegative bacteria was lower compared with Gram-positive bacteria. Indeed, the inhibition zones were in the range of 14– 17 mm and 15–21 mm for Gram-negative and Gram-positive bacteria, respectively. B. cereus, S. aureus and S. typhimurium were the most sensitive with halo diameters of 19, 21 and 17 mm, respectively. Furthermore, CBLS exhibited strong inhibitory activity against M. rouxii. At the same concentrations, the positive control bacitracin was found to have the strongest antibacterial activity against Gram-positive bacteria and the opposite trend was observed in the case of Gram-negative bacteria. These results were confirmed by the MICs (Table 6). In fact, the MICs are scientific and significant factors for evaluating the potential of this antimicrobial substance. This method could also compare naturally obtained antimicrobials with commercial antibiotics. The MICs of the produced CBLS ranged between 0.625 mg/mL and 5 mg/mL. Gram-positive bacteria were the most sensitive, with MICs of 1.25, 0.625 and 1.25 mg/mL for S. aureus, B. cereus and M. luteus, respectively. Compared with the positive control, CBLS was more effective against Gram-negative bacteria, especially against E. coli and S. typhimurium, with MICs of 1.25 mg/mL and 2.5 mg/mL, respectively. In the search for antibiotics produced by Bacillus spp., especially B. cereus, B. subtilis and B. licheniformis,
Table 6 Antimicrobial activity spectrum of the inhibitory substance. Indicator strain
Gram-negative bacteria Escherichia coli ATCC 25922 Pseudomonas aeruginosa ATCC 27853 Salmonella typhimurium ATCC 19430 Klebsiella pneumoniae ATCC 13883 Gram-positive bacteria Micrococcus luteus ATCC 4698 Staphylococcus aureus ATCC 25923 Bacillus cereus ATCC 11778 Enterococcus faecalis ATCC 29212 Fungi Aspergillus niger I1 Botrytis cinerea Mucor rouxii DSM 1191
An6 bacteriocin
Bacitracin
IZD (mm)
MIC (mg/mL)
IZD (mm)
MIC (mg/mL)
14 1.0
1.25
7 1.0
<5
17 2.0
2.5 9 1.0
<5
15 2.0 21 1.0 19 1.0
1.25 1.25 0.625 5
19 1.0 25 2.0 22 1.0 19 2.0
0.325 0.156 0.156 0.625
+
IZD, inhibition zone diameter; MIC, minimum inhibitory concentration. , no inhibition. Determinations were performed in triplicate and the data correspond to the mean standard deviation.
Please cite this article in press as: Ayed HB, et al. Isolation and biochemical characterisation of a bacteriocin-like substance produced by Bacillus amyloliquefaciens An6. J Global Antimicrob Resist (2015), http://dx.doi.org/10.1016/j.jgar.2015.07.001
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bacteriocin produced by Enterococcus mundtii, which inhibits the growth of Lactobacillus sakei [12].
Activity (U/ml)
2,4
OD 600 (nm)
50
2
40
1,6
30
1,2
20
0,8
OD (600 nm)
Activity (U/ml)
60
0,4
10
0
0 0
20
40 Time (h)
60
80
Fig. 2. Kinetics of bacteriocin-like substance production. OD600, optical density at 600 nm.
several antimicrobial compounds have been described [24]. Marahiel et al. [25] isolated a B. subtilis strain C126 from sugar cane fermentation that produces the polypeptide antibiotic bacitracin, which inhibited the growth of Micrococcus flavus. B. licheniformis strain 189 isolated from a hot spring environment in the Azores, Portugal, was found to strongly inhibit growth of Grampositive bacteria by producing peptide antibiotic [26]. 3.3. Pattern of antibiotic production by B. amyloliquefaciens An6 To study the production of antimicrobial compound during B. amyloliquefaciens An6 growth, the inhibitory activity present in cell-free samples taken at different time intervals was measured against the indicator strain S. aureus. Antimicrobial activity was detected at the mid-log growth phase and quickly reached a maximum at the early stationary phase (Fig. 2). After that, the antagonistic activity declined. This decrease may be attributed to adsorption on producer cells or to degradation by specific or nonspecific proteases [14]. The kinetics of this production was similar to those described for bacteriocins from Bacillus thuringiensis subsp. tochigiensis [22]. 3.4. Study of mode of action of antimicrobial compound produced by B. amyloliquefaciens strain An6 The mode of action of the CBLS was studied using S. aureus as the target bacteria. The inhibitory activity on the growth of S. aureus is shown in Fig. 3. Addition of CBLS to ca. 106 CFU/mL of indicator strain resulted in growth inhibition. Indeed, the OD600 of the treated culture changed significantly during the experiment. Cell lysis has been recorded when bacteriocin was added to actively growing cells of S. aureus, suggesting that the mode of action is bactericidal. A similar mode of action was reported for the 1
Control Bacteriocin added at 0 h Bacteriocin added after 2 h
OD (600nm)
0,8 0,6
CBLS
0,4
CBLS
0,2 0 0
2
4
6
8
5
10
Time (h) Fig. 3. Study of the mode of action of bacteriocin-like substance An6. OD, optical density; CBLS, crude bacteriocin-like substance.
3.5. Effects of enzymes, heat and pH on antimicrobial activity To determine the biochemical and biophysical properties of the inhibitory substance, samples were tested for sensitivity to enzymes, temperature and pH and the results are summarised in Table 7. Little or no change in the size of the inhibitory zone was observed when the same samples were treated with alcalase, trypsin, chymotrypsin, pepsin or catalase. There was a significant loss of inhibitory activity after treatment with proteinase K and catalase. The antimicrobial activity was sensitive to proteinase K and was unaffected by catalase activity, suggesting that it is proteinaceous in nature. These results suggest that this antimicrobial peptide possibly can survive in the intestinal environment and could be administered with feed. These results were different to bacteriocin-like substance produced by B. amyloliquefaciens isolated from the Brazilian Atlantic forest [27], which is sensitive to the proteolytic action of trypsin, papain, proteinase K and pronase E. The heat sensitivity of the antimicrobial substance was determined by measuring its activity following incubation for 15 min at different temperatures. It was stable at temperatures up to 80 8C but activity decreased at 100 8C and the substance was completely inactivated after incubation for 45 min at 121 8C. Lyophilisation and re-suspension of the sample did not affect the antimicrobial activity, indicating that this bioactive compound is attractive for potential biotechnological application. To evaluate the antimicrobial substance stability at different pH values, the CBLS was incubated at 25 8C at pH values of 4.0–10.0 for 2 h and activity was measured by the agar disk diffusion assay against S. aureus (Table 7). Activity was retained over a pH range of 4.0–10.0. These results were similar to coagulin, a bacteriocin-like inhibitory substance produced by Bacillus coagulans [28]. Bacteriocin An6, owing its wide activity spectrum and its high tolerance to acidic and alkaline pH values, temperature and proteases shows great potential for use as a food biopreservative. Food preservation has become a major issue because foodborne pathogens can cause havoc in preserved/fresh food items at high temperature, room temperature and even at low temperature. However, consumer demand for faster, healthier and ready-to-eat
Table 7 Effect of enzymes, heat and pH on antimicrobial activity. Treatment
Residual activity (%)a
Enzymes Proteinase K Trypsin Chymotrypsin Alcalase Pepsin
45 90 100 100 85
Heat/time 70 8C/15 min 80 8C/15 min 90 8C/15 min 100 8C/15 min 121 8C/45 min
100 100 95 70 –
pH 4 6 8 10
90 100 100 85
a Untreated crude bacteriocin-like substance (CBLS) was considered as 100% and the residual activity was defined as the ratio (treated/untreated CBLS).
Please cite this article in press as: Ayed HB, et al. Isolation and biochemical characterisation of a bacteriocin-like substance produced by Bacillus amyloliquefaciens An6. J Global Antimicrob Resist (2015), http://dx.doi.org/10.1016/j.jgar.2015.07.001
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of the abovementioned results, this antimicrobial substance shows a potential to be used in the medical and food industries. Stability under extreme pH values has an advantage for application in food as a biopreservative. Furthermore, heat resistance is an advantage since bacteriocins may remain active in foods after cooking and give protection against undesirable bacteria. Resistance to protease hydrolysis suggests that this antimicrobial peptide possibly can survive in the intestinal environment and could be administered with feed. Funding This work was funded by the Ministry of Higher Education and Scientific Research, Tunisia. Competing interests None declared. Ethical approval Not required. Fig. 4. Tricine–SDS-PAGE analysis and direct detection of antimicrobial activity. (A) Gel stained with Coomassie blue: lane 1, molecular weight marker; lane 2, crude supernatant. (B) The inhibition zone was shown by overlaying the gel with soft brain–heart infusion medium (0.75% agar, w/v) containing the Staphylococcus aureus indicator strain.
products has strongly demanded the use of more natural preservatives instead of chemical preservatives. Microbiologists became interested in bacteriocin-producing micro-organisms to overcome this problem that fulfil the requirement of food preservation. In this study, we have characterised a bacteriocinlike substance produced by B. amyloliquefaciens An6 strain that has a large inhibitory spectrum. This bacteriocin presents a wide spectrum of activity covering Gram-positive bacteria, Gramnegative bacteria and fungi. Considering the bacteriocin properties, its antimicrobial activity was stable over a wide temperature range from 70 to 100 8C and it was highly active over a wide range of pH from 4.0 to 10.0. Thus, this antimicrobial substance can be incorporated into food products to control microbial growth and to enhance the safety and shelf-life of food products. Heat stability is a very useful characteristic in the application of bacteriocin as a food preservative because many food-processing procedures involve a heating step. 3.6. SDS-PAGE analysis and mode of action of the inhibitory substance The dialysate generated after precipitation with ammonium sulphate was fractionated by ultrafiltration by using 10 kDa molecular weight cut-off membranes. Two fractions were obtained (concentrate and permeate). The antimicrobial activity was recovered in the concentrate, which contains peptides with a molecular weight >10 kDa. This results indicates that the active unit has a molecular mass >10 kDa. Direct detection of the antimicrobial activity was performed by Tricine–SDS-PAGE. As shown in the stained bands of the gel, several proteins were detected in the samples (Fig. 4A). Overlaying the gel with the indicator strain revealed a single protein band with inhibitory activity (Fig. 4B). The band had an apparent molecular mass of ca. 11 kDa. 4. Conclusion Here we report the production and characterisation of a bacteriocin-like substance from B. amyloliquefaciens. On the basis
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Please cite this article in press as: Ayed HB, et al. Isolation and biochemical characterisation of a bacteriocin-like substance produced by Bacillus amyloliquefaciens An6. J Global Antimicrob Resist (2015), http://dx.doi.org/10.1016/j.jgar.2015.07.001