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Journal of Applied Sciences Research, 4(11): 1315-1321, 2008 © 2008, INSInet Publication Purification and Characterization of Bacteriocin Produced by Isolated Strain of Lactococcus lactis 1 Hanaa A. El-Shafie, 2Nagwa Ibrahim, 2Hanem Abd El-Sabour and 1Yomna A. Mostafa. 1 Microbial Chemistry Department, National Research Centre, Cairo, Egypt. 2 Nutrition Department, Girl College, Ain Shams University, Egypt. Abstract: A bacteriocin-producing Lactococcus lactis isolated previously from traditional food. The bacteriocin termed nisin was purified to homogeneity by two-steps purification procedures including 70 % ammonium sulphate precipitation and cation exchange chromatography (DEAE cellulose). Using this procedure resulted in about 47.2 fold increase in specific activity and a recovery 26.4% of activity. The specific activity obtained was 9,917IU/mg when compared with the batch fermentation without recovery (2,105IU/mg). This bacteriocin has a bactericidal mode of action towards the tested spoilage and pathogenic strains. The Amino acid sequence determined for purified isolated strain exhibited high levels of similarity to the sequence of the reference strain. Results indicated excess of aspartic acid, valine, serine, proline and a deficiency of histidine, glycine, alanine, leucine, isoleucine in the isolated strain compared to the reference. NM R analysis revealed the presence of unsaturated amino acids dehydroalanine {DHA} and dehydrobutyrine {DHB}. Key word: Bacteriocins, Lactococcus lactis, nisin, bacteriocin purification INTRODUCTION In fermented foods, lactic acid bacteria (LAB) displays numerous antimicrobial activities. This is mainly due to the production of antimicrobial metabolites including organic acids, bacteriocins and antifungal peptides. Bacteriocin generally exert their antimicrobial action by interfering with the cell wall or the membrane of target organisms, either by inhibiting cell wall biosynthesis or causing pore formation, subsequently resulting in death [2 1 ]. Several bacteriocins with industrial potential have been purified and characterized.The highly promising results of these studies underline the important role that functional, bacteriocinogenic LAB strains may play in the food industry as starter cultures, co-cultures, or bioprotective cultures, to improve food quality and safety [4 ]. Lactococcus lactis strains are widely distributed used as starter for several types of cheese, fermented milk products,and ripened cream butter. T he fermentation of sugars, leading to a pH decrease which is important for milk clotting and reduction or prevention of adventitious microbial growth [1 5 ]. In the last years, the bacteriocins of lactic acid bacteria have attracted much attention because of their potential to increase safety and to extend shelf life of food [5 ]. Currently, only nisin has been Granted Generally Recognized As Safe (GRAS) status by the Food and Drug Administration (FDA). In addition,nisin used in combination with surfactants, chelators and adjuvants is also effective against both Gram -negative and resistant Gram-positive bacteria. Many lactic acid b a c teria p ro d uc e p ro teinaceo us antim icrob ials bacteriocins, some of which could provide valuable alternatives to traditional therapeutic antibiotics for the treatment of infectious diseases [ 1 9 ] . Two such bacteriocins, nisin and lacticin 3147, which are produced by strains of Lactococcus lactis are potential candidates. Nisin Z [1 7 ] and nisin Q [2 5 ] are two natural variants of the original nisin A, differing in their propeptide components from nisin A by one and four amino acids respectively. W e report here the purification and characterization of nisin , a novel nisin variant,by Lactococcus lactis. M ATERIALS AND M ETHODS M icroorganisms and Culture M edia: Lactic acid bacterial strain was isolated from yoghurt obtained from Cairo local market and identified in our laboratory as Lactococcus lactis[6 ]. Routine cultures were maintained on MRS media. Lyophilized reference strain Lactococcus lactis subsp.lactis NRRL B-1821obtained from the National Centre for Agricuture Utilization Research USA was grown and maintained on ATCC17 medium. Extraction and Purification of Nisin: One liter of Corresponding Author: Hanaa A. El-Shafie, Microbial Chemistry Department, National Research Centre, Cairo, Egypt. 1315 J. Appl. Sci. Res., 4(11): 1315-1321, 2008 MRS broth was inoculated with 1% (vol/vol) inoculum from an overnight MRS broth culture of Lactococcus lactis and incubated for 24 hr. at 37 o C. T he supernatant was separated by centrifugation (7000 *g for 30 min at 4 o C) , adjusted to pH 3.0, forwardly harvested with 60-80% ammonium sulphate. The supernatant containing unbound protein was discarded & the precipitated protein was collected by centrifugation, resuspend in 0.01 N HCl and dialyzed against 9 m Urea –HCl (pH 3.0). The bacteriocin was loaded on DEAE-cellulose sephadex gel filtration column, pre-equilibrated with the elution buffer (20 mM acetate buffer ,pH 3.6) .Finally bound bacteriocin was eluted with the same buffer at a flow rate of 1ml/min using a gradient of 0-1.2 NaCl. Active fractions were pooled ,evaporated, and the antimicrobial activity was assayed on each fraction. Nisin Unit: The titre in arbitrary units (AU) per milliliter is defined as the reciprocal of the highest dilution of the preparation for which a definite zone of inhibition is observed. Spectrum of Inhibitory Activity: The range of inhibitory activity of the isolated purified and the reference Lactococcus lactis (NRRL B-1821) strains were detected and compared with that of commercial nisin .The well diffusion direct assay described by Tagg and McGiven [1 1 ] and modified by Benkerroum & Sandine [1 ] was utilized to detect the inhibition of the sensitive strains.The GM 17 broth was supplemented with sodium B-glycerophosphate (2%) and catalase at final conc. of 100 U.20 ml of M 17 agar inoculated with 1ml of stationary phase sensitive indicator cultures( Table 1) was poured in sterile Petri dish and allowed to harden. Holes were punched out of the agar ,by using a cork bore (4mm of diameter ). The base of each hole was sealed with 50 ul of GM 17 soft agar (0.75%agar ) and then filled with 50 ul overnight test strains. The inoculated plates were incubated at 30 o C for 18 hr. and checked for the prescence of clear zones on inhibition as a result of antimicrobial activity. Solubility of Nisin: Solubility was determined using concentrated HCl solution of nisin (pH 2.0). Gradually increasing pH from 2-8, samples were equilibrated for 30 min under continous stirring and the nisin concentration was determined spectrometrically at 210nm [1 8 ]. Amino Acids Analysis: Mass spectrometry was conducted using Spectra –Physics Analytical, Inc. A 0099-600 with spectra. Focus optical Scanning detector and spectra UV 2000 and Ultrasphere C18 Beckman column. The analysis was carried out using a gradient of Pico-Tag solvent A&B at 40 o C and flow rate 1ml/min. Detection of the separated Pico-Tag-amino acids at 254 nm wave length. Before injecting of the sample, the illustrated was calibrated by two injections of the standard. Mass spectra were analyzed by using the manufacture"s software. NM R Analysis: A Jeol EX-270 NMR Spectrometer, 270 M Hz for H -was used with superconducting magnet from Oxford and 5mm Dual probehead for H- analysis. Typical conditions: Spectral width 4000 Hz&15000 Hz, 32 K data points and a flip angle of 450 [1 1 ]. RESULTS AND DISCUSSIONS Purification of the Inhibitory Peptide from Lactococcus lactis Strain: T he highest polypeptide activity produced by Lactococcus lactis was obtained by subjecting the supernatant to 70% ammonium sulfate fractionation. It was decided to proceed with the acidified extract of the cell pellet for purification purpose, since this comprised the majority of the inhibitory activity and was also of higher specific activity .The polypeptide was eluted effectively with purification fold 47.2 and 26.4% yield. The activity and purity of the inhibitory substance are shown in Table (2) and the elution pattern is presented in Fig. (1). Table 1: O rigin of Indicator Strains. Spoilage strains source Pathogenic strains Source Escherichia coli ATCC 25922 Clinical isolate Pseudomonas aeruginosa Local isolate ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Bacillus subtilis Food isolate Salm onella typhim urium Local isolate ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Bacillus m egaterium Food isolate Serratia m arcescens Clinical isolate ------------------------------------------------------------------------------------------Bacillus cereus Local isolate ------------------------------------------------------------------------------------------M ycococcus lactis Food isolate ------------------------------------------------------------------------------------------M icrococcus luteus Food isolate ------------------------------------------------------------------------------------------Staphylococcus aureus Food isolate 1316 J. Appl. Sci. Res., 4(11): 1315-1321, 2008 Table 2: Activity and purity of antibacterial substance produced by Lactococcus lactis during the fractionation procedure. Purification step Volum e Total activity Total protein Specific activity Recovery Purification (m l) (X103 IU ) (m g.) (IU /m g.) % fold Culture supernatant 900.0 7200.0 3420.0 2105.0 100.0 1.0 ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Precipitation with 70% 30.0 3095.0 330.0 9379.0 42.9 4.5 am m onium sulphate ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------D EAE-Cellulose 60.0 1904.0 19.2 9917.0 26.4 47.2 Fig. 1: Elution pattern of nisin produced by isolated strain Lactococcous lactis through DEAE-cellulose measured as protein concentration and activity. Antibacterial Spectrum: The isolated purified strain and the reference strain (Lactococcus lactis NRRL B-1821) were particularly broadly active in that they inhibited all the standard indicator strains as did the commercial nisin (Table 3). However, both strains are insensitive to commercial nisin. Solubility of Nisin: W ith respect to practical application, nisin solubility represents an important functional property. So solubility was determined over a wide pH range (2-8) and results are shown in Fig. (2). At low pH, the solubility of the isolated strain was 39mg/ml compared to the reference strain 40.8mg/ml. The solubility decreased with the increase in pH. Amino Acid Composition: The amino acid analysis of both strains the isolated purified and the reference are shown in Table (4). Results indicated excess of aspartic acid, valine, serine, proline and a deficiency of histidine, glycine, alanine, leucine, isoleucine in the isolated strain compared to the reference. However, unsaturated amino acids (dehydroalanine {DHA} and dehydrobutyrine {DHB}) are not determined (Figs. 3& 4). NM R Analysis: The chemical structure of the isolated purified antibiotic and the reference strain was investigated by H -NM R Spectroscopy. Results presented in Figs5&6 indicate the presence of two dehydroalanine (DHA) molecules and one molecule of dehydrobutyrine (DHB). These unsaturated amino acids characterized by the presence of vinyl protons which give resonance signals appeared as a peak. Results shows that the two vinyl protons of momolecular DHA give two peaks while the single vinyl proton of DHB give one peak. Discussion: Lantibiotics are a diverse group of heavily modified antimicrobial and/or signalling peptides produced by a wide range of bacteria, including a variety of LAB. Based on their diverse structures and mode of action, at least six separate antibiotic subgroups are characterized by significant post translational modifications, which include the formation of (B-methyl) lanthionines, among other unusual alterations. Since their discovery, interest has been fuelled by their obvious potential as food-grade antimicrobials to improve safety and quality, a potential which, to date, has been realized only by the longest characterized molecule, nisin [2 3 ]. Nisin, a bacteriocin secreted by Lactococcus lactis, is commercially used as a food preservative in forty Countries since 1983 [1 0 ]. The production of lantibiotics is widespread among gram-positive bacteria [2 0 ]. As more lantibiotics are detected, it is becoming increasingly clear that naturally occurring variants occur quite commonly,with some 1317 J. Appl. Sci. Res., 4(11): 1315-1321, 2008 Table 3: Activity spectrum of isolated and reference Lactococcus lactis strains. Indicator M icroorganism s M icroorganism s Sensitivity* ---------------------------------------------------------------------------------------------------------------------------------------------Isolated purified Lactococcus lactis Reference Lactococcus lactis Com m ercial nisin Escherichia coli + ++ ++ ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Staphylococcus aureus ++ +++ +++ ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Bacillus cereus +++ ++++ ++++ ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Bacillus subtilis ++ +++ +++ ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Bacillus m egaterium + + ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------M ycococcus lactis ++ ++ +++ ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------M icrococcus luteus ++ ++ ++ ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Pseudomonas aeruginosa + ++ ++ ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Serratia m arcescens + + ++ ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Salm onella typhim urium + + + Experim ental details: (-), not inhibited; (+), (++), (+++): low, high, very high inhibition, respectively. Fig. 2: Solubility of Nisaplain (commercial nisin), Nisin (Reference strain) and Nisin (isolated strain) in the pH range 2 to 8 at 25 O C. Table 4: Am ino acid com position of the purified peptide antibiotic from Lactococcus lactis (isolated strain and reference strain). Residue (% ) Reference strain Isolated strain Asparagin H istidine Glycin Alanine Valine Leucine Isoleucine Serin M ethionine Lysine Proline DHA DHB Lanthionine 7.5 3.8 0.3 2.3 2.8 6.1 33.0 4.3 16.0 11.0 6.6 N .D N .D N .D 8.5 2.0 0.2 1.3 3.7 5.2 29.0 4.8 15.6 10.0 10.4 N .D N .D N .D Total % 93.72% 90.70% of these differing in only a single amino acid residue (nisin A & nisin Z), whereas others involve several amino acid differences such as between epidermin and mutacin B-N y 266 [1 6 ]. It has been recommended that "by definition" natural lantibiotic variants should have only "a few "amino acid differences, essentially the same ring pattern, and that cross-immunity should be exhibited between their respective producing strains [2 6 ]. The bacteriocin was purified from the culture supernatant fluid of Lactococcus lactis to apparent homogeneity, as judged by two different protocols, 70% ammonium sulphate precipitation and cation exchange chromatography. The used protocols resulting in increase in specific activity and 26.4 % recovery (Table 1 & Fig 1). In accordance to our results Coventry et al[3 ] reported that, rude bacteriocin 1318 J. Appl. Sci. Res., 4(11): 1315-1321, 2008 Fig. 3: Amino acid composition of the purified peptide antibiotic from isolated strain. Fig. 4: Amino acid composition of the purified peptide antibiotic from reference strain. Fig. 5: NMR analysis of the peptide antibiotic purified from isolated strain. 1319 J. Appl. Sci. Res., 4(11): 1315-1321, 2008 including nisin prepared by ammonium sulfate precipitation. In Matsuski et al. [1 4 ] purified a peptide antibiotic from Lactococcus lactis IO-1 by simple procedure including acid treatment, ammonium sulfate precipitation, cation-exchange chromatography and reversed-phase high liquid chromatography and the specific activity of the peptide was 122 fold greater than the starting material and the recovery was 24% similar to our results. The relative low yield in the last step of the purification was most probably due to inactivation of the bacteriocin. The inhibitory spectrum of bacteriocin against s e v e r a l G r a m p o s itiv e a nd G r a m n e g a tiv e microorganisms is determined in order to evaluate the possibility of using the bacteriocin-producing strains as an additional barrier against spoilage and /or foodborne microorganisms in foods. The isolated purified Lactococcus lactis exhibited wide activity spectrum against pathogenic and spoilage microorganisms tested. These results are similar to Gupta and Batish [8 ]. The antimicrobial spectrum of the bacteriocin of ITAL 383 was similar to nisin of Lactococcus lactis subsp. lactis ATCC 11454. It showed a wide spectrum of inhibitory activity affecting closely related species and all other Gram-positive microorganisms examined. The bacteriocin did not inhibit Bacillus megaterium. This inability of the bacteriocin over some microorganisms was previously reported [2 ,1 2 ,2 4 ]. Solubility of nisin over a wide range of pH (2.0-8.0) has been evaluated. It has been noted that the solubility decease directly with the decrease of pH. Hurst [1 1 ] and Liu & Hansen [1 3 ] stated that nisin solubility depends strongly on the pH. The sequence of the amino acids residues determined for the purified Lactococcus lactis (aspartic-serine-glycine-histedine-alanine-proline-valinemethionine-leucinelysine) exhibited high levels of similarity to the sequence of the reference strain (aspartic - serine - glycine - alanine- proline - valine methionine - isoluecine - leucine). Also our results referred to the presence of an excess of aspartic acid and the deficiency of histidine in the isolated strain being similar to nisin Z. However, failure of appearance of unsaturated amino acids (DHA & DHB) may be due to acid hydrolysis of the purified peptide as reported by Gross & Morell[7 ]. NMR analysis of the purified peptide and the reference revealed that the tested samples may have two DHA molecules and one DHB molecule, because these unsaturated amino acids characterized by the presence of vinyl protons which give resonance signals appeared as peaks. 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