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Isolation and Purification of Two Bacteriocins 3D Produced by Enterococcus faecium with Inhibitory Activity Against Listeria ....
Article in Current Microbiology · February 2011
DOI: 10.1007/s00284-010-9732-0 · Source: PubMed
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Isolation and Purification of Two Bacteriocins 3D Produced by Enterococcus faecium with Inhibitory Activity Against Listeria monocytogenes
Kaoutar Bayoub•Ilham Mardad• Emna Ammar• Aurelio Serrano• Abdelaziz Soukri
Received: 9 October 2009 / Accepted: 13 July 2010 / Published online: 18 August 2010 ÓSpringer Science+Business Media, LLC 2010
Abstract
Strain 3D, isolated from fermented traditional Moroccan dairy product, and identified as Enterococcus faecium, was studied for its capability to produce two bacteriocins acting against Listeria monocytogenes. Bac- teriocins 3 Da and 3Db were heat stable inactivated by proteinase K, pepsin, and trypsin but not when treated with catalase. The evidenced bacteriocins were stable in a wide pH range from 2 to 11 and bactericidal activity was kept during storage at 4°C. However, the combination of tem- perature and pH exhibited a stability of the bacteriocins.
RP-HPLC purification of the anti-microbial compounds shows two active fractions eluted at 16 and 30.5 min, respectively. Mass spectrometry analysis showed that E. faecium 3D produce two bacteriocins Enterocin 3 Da (3893.080 Da) and Enterocin 3Db (4203.350 Da). This strain is food-grade organism and its bacteriocins were heat-stable peptides at basic, neutral, and acid pH: such bacteriocins may be of interest as food preservatives.
Keywords
Bacteriocins Antimicrobial activity Enterococcus faecium Listeria monocytogenes Mass spectrometry Food preservatives
Introduction
In spite of the use of modern technologies in food pro- duction and the implementation of quality standards, food poisonings are the cause of 6.5 to 33 million human dis- eases and more than 9000 death/year in the world [24].
Bacterial fermentation of perishable raw materials has been used for centuries to preserve the nutritive value of food and beverages over an extended shelf-life. In a number of food fermentations, the key event is the con- version of sugars to lactic acid by lactic acid bacteria (LAB, which include the genera Lactococcus, Streptococ- cus, Lactobacillus, and Pediococcus, among others).
These bacteria are among the most well-known and investigated producers of microbial antagonists. They are of particular interest in terms of the widespread occurrence of bacteriocins within the group and are also in wide use throughout the fermented dairy-, food-, and meat-process- ing industries. Their role in the preservation and flavor characteristics of foods has been well documented [7,
22].Many lactic acid bacteria have important roles in the production of fermented foods. They are the most com- monly given generally recognized as safe (GRAS) status, and they have the potential to control food-borne pathogens and to extend the shelf life of food [2,
19,30].Enterococci are part of the natural intestinal flora of humans and animals and play an important role in main- taining the microbial balance [21]; they are also promising for the biopreservation of food, especially by means of bacteriocin production [13]. Bacteriocins are ribosomally synthesized antimicrobial peptides with activity that is usually directed against species closely related to the pro- ducing bacterium [18,
20].The bacteriocins, antibacterial proteinaceous substances, are suitable candidates for using as natural preservative of
K. BayoubI. MardadA. Soukri (&)Laboratory of Physiology and Molecular Genetics (PGM), Faculty of Sciences, Department of Biology, Aıˆn chock, University Hassan II, Casablanca, Morocco
e-mail: a_soukri@hotmail.com E. Ammar
Department of Biological Engineering/LARSEN, National Engineering School in Sfax, Sfax, Tunisia
A. Serrano
Instituto de Bioquı´mica Vegetal y Fotosı´ntesis, Centro de Investigaciones Cientı´ficas, CSIC-Universidad de Sevilla, Seville, Spain
DOI 10.1007/s00284-010-9732-0
food, able to inhibit the growth of some food-borne bac- teria [8,
15].Furthermore, as the majority of bacteriocin-producing LAB are natural food isolates, they are ideally suited to food applications. Although various methods other than bacteriocin production are employed for the preservation of food/beverages, an increasingly, health conscious public may seek to avoid foods that have undergone extensive processing or which contain chemical preservatives.
Therefore, the production of bacteriocins by LAB is not only advantageous to the bacteria themselves but could also be exploited by the food industry as a tool to control undesirable bacteria in a food-grade and natural manner, which is likely to be more acceptable to consumers.
The classic example of a commercially successful nat- urally produced inhibitory agent is nisin. Known since 1928 to be produced by some Lactococcus lactis isolates and structurally characterized in 1971 as a lanthionine- containing peptide, nisin and nisin-producing strains have a long history of application in food preservation, especially of dairy products [19].
Much of the research on the application of class IIa bacteriocins has focused on the use of the bacteriocin producing culture in foods to control the growth of spoilage organisms or food-borne pathogens, such as L. monocyt- ogenes [12].
The aim of this study was to detect and characterize the bacteriocins produced by E. faecium 3D isolated from fer- mented traditional Moroccan dairy products. Such products were selected based on their antagonistic effect against Lis- teria monocytogenes. These substances were characterized, and an approach of purification was considered allowing biotechnological applications of these products.
Materials and Methods
Bacterial Strains and Growth Conditions Bacterial Isolation
Enterococcus faecium 3D was obtained from fermented traditional dairy product (raıˆb; a traditional Moroccan curdled raw milk by spontaneous fermentation) which was serially diluted with sterile saline solution (0.9% NaCl (w/v)). Aliquots of 0.1 ml were spread onto plates of MRS agar (Man, Rogosa, Sharpe-Biokar, diagnostics, France).
The plates were then incubated at 30°C for 24 h.
Screening for Bacteriocin Production
Listeria monocytogenes ATCC 19117 was used as an indicator strain in assays of antimicrobial activity. The
indicator strain was cultured for 18 h at 37°C and main- tained at TSB-YE (Tryptic Soy Broth-Yeast extract—
Biokar, diagnostics, France).
Briefly, cultures were streaked onto MRS agar plates and incubated for 24 h at 30°C. After incubation, the col- onies were picked off and stabbed onto fresh MRS agar plates. The fresh plates were incubated at 30°C for 16 h, and then 5 ml of TS-YE soft agar (0.7%, w/v) containing about 10
6cfu of L. monocytogenes ATCC 19117 poured over the plates. After incubation at 30°C for 24 h, the plates were checked for inhibition zones.
Colonies of lactic acid bacteria with the largest inhibi- tion zones were selected and purified by streaking the bacteria onto MRS agar. The presence of antimicrobial compounds in cell-free culture supernatants was confirmed by using the agar spot method [32].
All cultures were routinely stored at 4°C and maintained as frozen stocks at
-20°C in 40% glycerol, propagated andincubated twice for 18 to 24 h at 30°C in MRS broth media before experimental use.
Bacterial Identification
The bacterial strain was identified through morphological, cultural, and genetic analyses. The 16S rDNA gene was sequenced.
For genotypic identification, total DNA was extracted from bacterial cultures grown to stationary phase using the Wizard Genomic DNA isolation kit (Promega) according to the manufacturer’s instructions and was used for PCR amplification.
Homologies were searched in the BLASTN database (National Center for Biotechnology Information) using BLAST.
Antibacterial Activity Assay
To assay the inhibitory activity of the bacteriocin and to study their physico-chemical properties, the agar-well dif- fusion method Schillinger and Lucke, [31] was employed.
Ten ml of agar medium (0.7% w/v) was inoculated with 0.1 ml of fresh overnight culture of the indicator strain and poured into a petri dish. Wells of 5 mm diameter were performed in the agar medium and filled with 50
ll of thesample. The plates were incubated for 18 h at 30°C, and then examined for inhibition of the bacterial growth. The diameters of the inhibition zones were measured.
Inhibitory Spectrum of the Bacteriocin
Cell-free culture supernatants obtained by centrifugation of cultures at 10000 g at 4°C for 10 min were adjusted to
480 K. Bayoub et al.: Isolation and Purification of Two Bacteriocins
pH 6 with 1 M NaOH, filtered through 0.45
lm pore-sizefilters. The antimicrobial activity of the supernatant was determined by the spot-on-lawn method [32]. The spectrum of antimicrobial activity was determined by screening against the strains listed in Table
1. A clear inhibition zoneat least 2 mm in diameter was recorded as positive.
Growth and Bacteriocin Production Kinetics
MRS broth (200 ml) was inoculated with 1% of an over night culture of strain 3D and incubated at 30°C without agitation for 30 h. At appropriate intervals, samples were removed for measurement of biomass by absorbance at 660 nm.
Stability against Enzymes, Heat, and pH
The cell-free culture supernatant containing bacteriocin was treated with proteolytic enzymes, heat, and different pH
values in order to determine the level of its sensitivity to these factors. After being treated, the samples were tested against L. monocytogenes by the agar-well diffusion method.
Effect of Enzymes on Bacteriocin Activity
The chemical nature of the antimicrobial substance produced by this strain was investigated by determining its sensitivities to different enzymes. The cell-free culture supernatant was treated with catalase (SIGMA) and three different proteo- lytic enzymes (SIGMA): Proteinase K, Pepsin, and trypsin.
The enzymes were filtered through 0.22
lm filters (Milliporefilters, Schleicher & Schuell) and added to cell-free culture supernatant allowing a final concentration of 1 mg/ml. The solution was incubated at 37°C for 2 h. In the same condi- tions of incubation, an untreated sample was used as the positive control.
Effect of pH on Bacteriocin Activity
To test pH stability, the samples were adjusted to pH 2, 4, 5, 6, 7, 9, 10, and 11 with 5 M NaOH and 5 N HCl. The MRS broth (pH 2–11) was used as the control.
Effect of Temperature on Bacteriocin Activity
To determine heat stability, the samples were heated to 121°C for 15 min, 100°C for 20 min, and 60°C for 60 min, and then the samples were tested for bacteriocin activity.
Effect of pH Combined with the Heat Treatment on Bacteriocin Activity
To study the sensitivity of bacteriocin to pH combined with the heat treatment, aliquots of 1 ml cell-free culture supernatant were adjusted to pH 2, 4, 5, 6, 7, 9, 10, and 11 and then heated at 121°C for 20 min. Subsequently, the samples were cooled to room temperature before being tested for inhibitory activity.
Mode of Action
Prior to the experiments on the mode of action, the bac- teriocin arbitrary unit (AU), defined as the reciprocal of the highest dilution of culture supernatant giving an inhibition zone in the agar well diffusion test against indicator strain, was determined in the cell-free supernatant considered (i.e., the crude bacteriocin). Then, the crude bacteriocin (640 AU/ml) was added to cultures of the indicator strain L.monocytogenes after 3 h of growth. Bacterial growth was monitored by measuring the optical density at 600 nm at different time intervals.
Table 1 Strains used in this study for spectrum determination of antimicrobial activity, with their cultivation media and diameter of inhibition zone
Indicator strains Media Inhibition
zone (mm) Listeria monocytogenesATCC 19117 TSB-YE 25 Enterococcus fecalisATCC 29212 MRS 10 Lactobacillus bulgaricus340
(Rhodia Food-France)
MRS 14
Salmonella curvalisRVA 1876 (IP, Tunis) NB 8 Salmonella curvalisRV3 3935 (IP, Tunis) NB 9 Salmonella curvalis
RVA 06 3607 (IP, Tunis)
NB 9
Salmonellaspp.44846 (IP, Tunis) NB 8
Salmonellaspp. 780 (IP, Tunis) NB 8
Salmonellaspp. 3631 (IP, Tunis) NB 9 Salmonellaspp. 2687 (IP, Tunis) NB 8 Salmonella typhimuriumNRRL-B 4420 NB 9
Escherichia coliATCC 25922 NB 2
Staphylococcus aureusATCC 25923 NB 3
Micrococcus luteusNCIMB 8166 NB 8
Pseudomonas aeruginosaATCC 27853 NB 5
Serratia marcescurs(C.H.U) NB 0
Klebsiella pneumoniae(C.H.U) NB 0
Enterobacter cloacae(C.H.U) NB 0
Acinetobacter baumannii(C.H.U) NB 0
ATCCAmerican type culture collection,NRRL-B: ARS culture col- lection, Northern Regional Research Laboratory, NCIMB National Collection of Industrial and Marine Bacteria, IP: Institut Pasteur, Tunis, Tunisia, C.H.U Center Hospital University IBN ROCHD, Casablanca, Morocco, TSB-YETryptic Soy Broth–Yeast extract,MRS Man, Rogosa, Sharpe,NBnutrient broth
Purification of the Inhibitory Substance from E. faecium 3D
The inhibitory substance of E. faecium 3D was purified from 200 ml cultures. MRS broth was inoculated with the strain for 18 h at 30°C. Cells were removed by centrifu- gation (100009g at 4°C, 10 min) and the peptide precipi- tated from the cell-free supernatant with 80% saturated ammonium sulfate. The precipitate was resuspended in phosphate buffer (pH 6.8, 20 mM), desalted overnight against 2 l of the same buffer using dialysis membrane (Spectra. MW cut-off: 1000 Da) and loaded on a Sep-Pak C18 cartridge (Water Millipore, MA, USA). The column was initially hydrated with acetonitrile 100%, then washed successively with 5 ml of 0, 10, 20, 30, 40, 60, 80, and 100% of solvent B [Isopropanol/acetonitrile 2:1] in solvent A [0.1% trifluoroacetic acid (TFA)]. All eluted fractions were separately concentrated under vacuum and tested for antimicrobial activity. The anti-Listeria fractions were eluted with 40 and 80% [Isopropanol/acetonitrile 2:1]. The active fractions were pooled and dissolved in 0.1% (v/v) TFA. This last fraction was then purified by two HPLC steps (LaChrom ELITE VWR Hitachi, with a 5
lm C18VYDAC column). After equilibration of the C18 column with water/TFA 0.1% (v/v), at a flow rate of 1 ml min
-1, peptides were eluted by increasing the concentration gra- dient of this solvent (0.1% TFA in acetonitrile) as follows:
0–5 min: 20% (v/v) acetonitrile; 5–35 min: 20–100% (v/v) acetonitrile; 35–40 min: 100% (v/v) acetonitrile. Peptides were detected spectrophotometrically by measuring the optical density at 220 nm. Fractions corresponding to all peaks were collected independently, concentrated under vacuum and assayed for antimicrobial activity.
Tricin-SDS Polyacrylamide Gel Electrophoresis
Tricin SDS–PAGE was carried out according to the method of Schagger and Jagow [29]. The gel contained 4 and 16.5% polyacrylamide in the ‘‘stacking’’ and ‘‘separating’’
gels, respectively. After electrophoresis, the gel was divi- ded into two parts: the first half, including both standards and bacteriocin sample, was stained with Coomassie Bril- liant Blue R-250, and the second part was used for activity detection according to the method of Bhunia and Johnson [4]. The gel unstained was placed in sterile distilled water for 1 h, and then overlaid with the overnight indicator strain.
Mass Spectrometry
The lyophilized fractions were analyzed with the mass spectrometer model Autoflex Bruker. The MALDI-TOF system (matrix-assisted laser desorption ionization time-of-
flight) was used with a matrix type
a-cyano-hydroxycin-namic acid.
Results and Discussion
Strain Identification
Strain 3D was selected for its antimicrobial ability against the pathogenic germ L. monocytogenes ATCC 19117. It was identified as a member of the genus Enterococcus based on following criteria: cocci, gram-positive, catalase negative, oxidase negative and ability to grow at 10 and 45°C, in the presence of 6.5% NaCl, at pH 9. This iden- tification was confirmed by 16S DNA sequence analysis.
The sequence obtained was 454 bp, it was compared to the databases using BLAST program through the National Center for Biotechnology Information (NCBI), and revealed a similarity of 99% with E. faecium 3D.
Inhibitory Substances Production Kinetic
Antibacterial compounds secreted by E. faecium 3D was produced during the exponential growth phase of batch culture. The highest bacteriocin production level was noted during the beginning of the stationary phase, where the antibacterial concentration reached 64000 AU/ml. This level remained stable during all this phase. This behavior regarding bacteriocin production is well-known and was described in previous studies [1,
10,11,26].Spectrum of Inhibition of the Bacteriocin
Bacteriocins from E. faecium 3D were active against a wide range of bacteria (Table
1), including food-bornepathogens and spoilage bacteria: Enterococcus fecalis, Staphylococcus aureus, Micrococcus luteus, Lactobacillus bulgaricus, Salmonella curvalis, Salmonella spp. Salmo- nella typhimurium, Escherichia coli, Pseudomonas aeru- ginosa. The strain E. faecium 3D was highly active against L. monocytogenes. The same spectrum of activity was obtained with partially purified bacteriocins 3D.
Effects of pH, Temperature, and Enzymes on Bacteriocin Activity
To characterize the chemical nature of inhibitory substance produced by strain selected E. faecium 3D, a study of the stabilities of active substance samples was carried out in the presence of proteolytic enzymes and catalase and changes in heat and pH. The results (Table
2) showed thatthe inhibitory substance was highly stable up to 120°C for 20 min and from pH 2 to 11. The effect of various enzymes
482 K. Bayoub et al.: Isolation and Purification of Two Bacteriocins
on the inhibitory agent was studied. Complete inactivation was observed after treatment of the cell-free supernatant with proteinase K, trypsin, pepsin which indicated the proteinaceous nature of the active agent.
The antibacterial activity of the bacteriocins tested remained preserved while freezing cell-free culture super- natants at
-20°C during 10 months. Moreover, the bacte-riocin was active under different pH values, ranging from 2 to 11, with a maximum activity at pH 6 and reduction in activity of approximately 25% under pH 11 (data not shown). This indicates that such bacteriocins may be useful in acidic as well as non-acidic foods. These results are in agreement with several bacteriocins from enterococci strains and others genus of strains; enterocin EL1 [23], bacteriocin produced by Streptococcus thermophilus 81 [17], nisin-like bacteriocin [5], bacteriocin ST 15 [9]. The activity of the studied bacteriocins was not affected by heat at 121°C for 20 min; they were resistances to autoclaving conditions in low pH.
Effects of pH Combined with the Heat Treatment on Bacteriocin Activity
The temperature and the pH are two parameters influencing the stability of the bacteriocins. The combined effect of these two factors was studied on the experimented bacteria.
E. faecium 3D had activity in a broad zone of pH from 2 to 11. After heat treatment for each pH value studied, the stability of the bacteriocins showed some resistance to autoclaving at low pH, however the activity was lost at pH values above 6.0 (Fig.
1). Both the culture supernatant andthe partial purified bacteriocins fraction exhibited the same mentioned results. From these results we can suggest that the structure of our bacteriocins is resistant to the auto- claving, and its activity is stable at low pH combined to
heat treatment. This could be interesting as regards man- ufacturing and sterilizing food that has an acidic pH. Our results were in accord with those found by Ryan et al. [27], who showed that lacticin 3147, a bacteriocin produced by L. lactis DPC3147 is thermostable in particular at acidic pH. Similarly, Choi et al. [5] showed that the bacteriocin nisin-like produced by L. lactis subsp. Lactis A164 is thermostable particularly under low pH.
Mode of Action
Addition of E. faecium 3D bacteriocins to L. monocytogenes after 3 h of growth resulted in growth inhibition (Fig.
2),suggesting that the mode of action is bacteriostatic.
Purification of the Bacteriocins and Molecular Weight Determination
Results of the various purification steps are given in Table
3.A three-step protocol has been developed for the puri- fication of bacteriocins from E. faecium 3D cultures taken
Table 2 Effect of proteolytic enzymes, pH, and temperature on theactivity of bacteriocins 3D
Treatment Activity
Enzymes (1 mg/ml)
Proteinase K –
Pepsin –
Trypsin –
Catalase ?
pH
2–11 ?
Temperature
60°C for 60 min ?
100°C for 20 min ?
121°C for 15 min ?
-no inhibition activity
?inhibition activity
Fig. 1 Sensitivity of E. faecium 3D bacteriocins to pH combined with the heat treatment (121°C-20 min)
0 0,2 0,4 0,6 0,8 1 1,2
0 1 2 3 4 5 6 7
Time (h)
OD (600 nm)
Control Treated with E. faecium 3D bacteriocins Bacteriocins
addition
Fig. 2 The effect ofE. faecium3D bacteriocins (6400 AU/ml) on the growth ofL. monocytogenes. Growth ofL. monocytogeneswithout bacteriocins (filled square) and with bacteriocins (filled triangle). The arrowshows the addition of bacteriocins
at the exponential growth phase. The cell-free supernatant was subjected to ammonium sulfate precipitation. The next purification step consisted in loading on a Sep-Pak C18 column. This step showed the hydrophobic nature of bac- teriocins from E. faecium 3D. The most active fractions were pooled, concentrated under vacuum, and subjected to C18 RP-HPLC. The active fractions were collected and re- injected onto the RP-HPLC. The final RP-HPLC step allowed elution of two separate active fractions eluted at 16 and 30.5 min, respectively (Fig.
3).Based on activity measurement (Table
3), 80% of thebacteriocins activity present in the cell-free supernatant
was recovered after ammonium sulphate precipitation, and it was further purified on a Sep-Pak C18 column. The Sep- Pak C18 restored only 0.125% of the initial culture supernatant. Final purification of bacteriocins 3D by reverse-phase HPLC revealed the presence of two distinct peaks eluted at 46 and 88% respectively, corresponding to retention times of 16 and 30.5 min, respectively (Fig.
3).Analysis of the purified bacteriocins produced by E. faecium 3D by Tricine-SDS–PAGE, and direct detection of antimicrobial activity on the electrophoresis gel indi- cated two bands that apparent molecular masses were estimated to be between 4 and 7 kDa (Fig.
4). However,several authors noted that the migration of small, hydro- phobic peptides in SDS–PAGE did not correlate with their true size [3,
14, 25]. Purified bacteriocins 3D were sub-jected to mass spectrometry analysis giving two molecular masses of 3893.080 and 4203.350 Da. That confirms the result given by RP-HPLC. These results are close to that reported for enterocin Q (3970.31 Da) [6] and enterocin CRL35 (4308.55 Da) [28], but smaller than the bacterio- cins from E. faecium MMT21: enterocin A (4828 Da) and B (5463 Da), respectively [16].
Table 3 Purification steps of anti-Listeriapeptides produced byE. faecium3D
Purification step Volume (ml) Activity (AU/ml) Total activity (AU) Yield (%)
Crude extract (culture supernatant) 200 649103 128009103 100
Ammonium sulphate precipitation 10 10249103 102409103 80
C18 Sep-Pak eluate 1 169103 169103 0.125
C18 RP-HPLC eluate 0.5 89103 49103 0.03125
Fig. 3 Reverse-phase HPLC profile of the active fractions. The inhibition zones of the active peaks are shown in the figure. Fraction AEnterocin 3Da and fractionBEnterocin 3Db
Fig. 4 Direct detection of pure bacteriocins activity fromE. faecium 3D on agar plate after Tricine SDS–PAGE
484 K. Bayoub et al.: Isolation and Purification of Two Bacteriocins
Conclusion
From this study, it may be concluded that the antimicrobial peptides were heat stable and were not sensitive to acid and alkaline conditions (pH 2–10), but they were sensitive to several proteolytic enzymes. Their inhibitory activity was completely eliminated after treatment with proteinase K, trypsin, and pepsin. The broad spectrum of antimicrobial activity, and its resistance to pH and heat, renders bacte- riocins 3D the good candidate as a natural fermented food preservative.
Acknowledgments The authors would like to express their grati- tude to Mr. El Mzibri, biology and medicals researches unity-CNE- STEN (Centre National de l’Energie, des Sciences et des Techniques Nucle´aires), for the molecular identification of the strains.
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