This action might not be possible to undo. Are you sure you want to continue?
Application of Biotechnology for Production, Purification and Characterization of Peptide Antibiotic Produced by Probiotic Lactobacillus plantarum, NRRL B-227
H.M. Atta, B.M. Refaat and A.A. El-Waseif Botany and Microbiology Department Faculty of Science (Boys) Al-Azhar University, Cairo, Egypt
Abstract: This work was carried out in the course of a screening program for specification of the bioactive substances that demonstrated inhibitory affects against bacterial pathogens and application of oral administration of probiotic Lactobacillus plantrum NRRL B-227 in vivo using mices. Forty lactobacillus sp. selected from American agriculture culture collection. All these strains were screened for their antibacterial activity against pathogenic bacteria. Among the forty Lactobacillus, the broad spectrum strain Lactobacillus sp NRRL B-227 was active against bacterial pathogens, Gram positive bacteria viz Staphylococcus aureus ATCC 29213, Micrococcus luteus ATCC 9341, Bacillus subtilis NRRL B-543, Bacillus subtilis NCTC 10400, Bacillus pumilus NCTC 8214 and Gram negative bacteria viz Escherichia coli ATCC 25922, Escherichia coli NCTC 10416, Salmonella typhi NCIMB 9331, Enterobacter cloacae ATCC 13047, Pseudomonas aeruginosa ATCC 10145, Klebsiella pneumonia NCTC 9111, Klebsiella pneumonia ATCC 13883. From the taxonomic features, the Lactobacillus sp NRRL B-227 matches with Lactobacillus plantarum in the morphological, physiological and biochemical characters. Thus, it was given the suggested name Lactobacillus plantarum NRRL B-227. The nucleotide sequence of the 16S RNA gene of the most potent strain evidenced a 100% similarity with Lactobacillus plantarum. The active metabolite was precipitation using ammonium sulfate and its purification was performed using reverse-phase high performance liquid chromatography (RP-HPLC) on ODS Hypersil C18 column. The physico-chemical characteristics of the purified peptide antibiotic viz. heat & pH resistance and spectroscopic characteristics (UV, IR, Mass spectrum and amino acid analyzer) have been investigated. In conclusion, the collected data emphasized the fact that the purified peptide antibiotic may be belonging to bacteriocin antibiotic produced by Lactobacillus plantarum NRRL B-227. The application of oral administration of probiotic Lactobacillus plantrum NRRL B-227 in vivo using mice's had been investigated. Key words: Lactobacillus plantarum NRRL B-227 % Antibacterial activity % Bacteriocin % 16S RNA % Fermentation % Precipitation and Purification % Probiotic Lactobacillus plantarum in vivo INTRODUCTION Lactic acid bacteria have been widely used in the food industry as starter culture for fermentation. Lactobacillus species play a crucial role in foodstuffs, because of their fermentative ability and their health and nutritional benefits . Lactic acid bacteria can produce many compounds such as organic acids, hydrogen peroxide and bacteriocin during fermentation [2,3]. Bacteriocins are proteinaceous substances that display antimicrobial activity against species closely related to the producer strain and/or other bacteria [4,5]. A variety of antimicrobial agents that differ in their inhibitory spectra, mode of actions and biochemical characteristics is produced by Lactobacillus species. L. plantarum has been isolated from various habitats and several bacteriocins (antimicrobial peptides) have been described in strains from milk and cheese [6,7]. Bacteriocins are proteinaceous antibacterial compounds and exhibit bactericidal activity against species closely related to the producer Lactobacillus strains [8, 9]. Many bacteriocins are active against food-borne pathogens, especially against Listeria monocytogenes [10, 11]. Bacteriocins are heterogeneous and they are classified largely based on differences in their molecular weights . Some bacteriocins are peptides consisting of only 13 to 37 amino acids. Some small bacteriocins contain unusual amino acids originating from modifications of conventional amino acids after translation . Several types of bacteriocins from food-associated lactic acid
Corresponding Author: Houssam M. Atta, Department of Botany and Microbiology, Faculty of Science, (Boys) Al-Azhar University, Cairo, Egypt
Primers used for PCR and DNA sequencing. NRRL B-227 selected from American agriculture culture collection.15]. 2 g Dipotassium hydrogen phosphate. Bacterial Identification: Selected strain was examined microscopically for cellular morphology and biochemical properties . Probiotic lactobacilli are known to confirm an array of health promoting activities on their host after either parenteral or oral administration . It has been reported that nisin is more active against Gram-positive bacteria.microbial activity was determined by cup method assay according to . Bacillus subtilis NRRL B-543.). physiology and biochemical characteristics. The PCR amplification was performed with the primer pair SPO/SP6 targeted against regions of 16S ribosomal DNA StrepF 5AAGAGTTTGATCCTGGCTCAG-3. Final pH 6. Klebsiella pneumonia NCTC 9111. Phylogenetic analysis was realized by an alignment of sequence consensus of the 16S ribosomal DNA genes collected in an international database (Gene bank). Biotech.Global J. 21]. bacteriocin and nisin produced by Lactococcus lactis sp. purification and the physico-chemical characteristics have been investigated. 4 (2): 115-125. 8 g Meat extract. USA) and sequenced using the Big Dye Terminator V3. 2 g Triammonium citrate. USA). has been the most extensively characterized [16. water. Screening for Antibacterial Activity: The anti. The current work aimed to a screening program for specification of the bioactive substances produced by Lactobacillus sp. 16S ribosomal DNA PCR applicants were purified following the microcon YM-100 kit (Bedford. The results were then expressed in percentage of homology between the submitted sequence and the sequences resulting from the database . production of inhibitory compounds and rebalancing of disturbed gastrointestinal microbial composition and metabolism . Madison. polymerisation at 72°C (2 min. The identification of this strain. Bacillus subtilis NCTC 10400 and Gram negative bacteria viz Escherichia coli ATCC 25922. 1L Dist. Of these. Micrococcus luteus ATCC 9341. diplococcin. and Strep R 5CTACGGCTACCTTGTTACGA-3 . However. 5 g Sodium acetate trihydrate. helveticins. PCR conditions included a hot start at 96°C (5 min. MATERIALS AND METHODS Bacterial Culture and Media: Bacterial strain used in this study were selected from culture collection NRRL B. acidophilin. 0. PCR products were resolved by electrophoresis in 1% (w/v) agarose gel and visualized by ethidium bromide (1 µl/10 ml) staining. based on the cultural. Salmonella typhi NCIMB 9331. Estimation of Total Protein: The protein content of the peptide antibiotic was determined according to .). Nisin is the only bacteriocin commercially available . Some of their beneficial effects include prevention of intestinal infection  anticarcinogenic activity  control of serum cholesterol and enhancement of immunity  growth enhancement of animals . 0.17]. & Biochem. Other bacteriocins of Lactobacilli have been reported to be effective against closely related species of mesophilic Lactobacillus and therefore considered as potential natural food preservatives [20. lactacins. of which nisin.227 grown in MRS broth (10 g Peptone. lactolin and plantaricins are the important bacteriocins [14. Bacillus pumilus NCTC 8214. 35 cycles consisting of hybridation at 50°C (1 min). Klebsiella pneumonia ATCC 13883. The bioactive substance was precipitation. bulgarican. as well as 16s rRNA methodology. PCR was used to amplify the 16S ribosomal DNA gene of strain. MA. 116 Indicator Bacterial Strains: Gram positive bacteria viz Staphylococcus aureus ATCC 29213. lactis. 20 g D(+) Glucose.2 ) at 30°C for 48 h . which generates an antibacterial compound. The 16S ribosomal DNA sequence was determined by direct sequencing. particularly the spore-formers .0 kit as specified by the supplier with primers while automated sequencing of both strands of the PCR products gene sequencer (ABI. Escherichia coli NCTC 10416. USA). studies relating to the antibacterial properties of these organisms are limited and not fully exploited for use [22. morphology. 23]. denaturation at 96°C (1 min) and a final extension at 72°C (2 min. 4 g yeast extract.05 g Magnesium sulfate tetrahydrate. The sequences obtained (500-750 bp) were then assembled in silico (Vector NTI) using overlapping zones between the various sequences to form the contiguous sequence.. The mechanism by which these probiotics affects their host and bring about improvement in the gut barrier can be due to competition for adhesion site. is also reported.). Pseudomonas aeruginosa ATCC 10145. Forster. Total DNA was isolated by using Wizard genomic DNA purification kit (Promega. Enterobacter cloacae ATCC 13047. 2009 bacteria have been identified and characterized. Amplification of DNA was performed in a Mastercycler personal thermal cycler (Eppendorf). Application of oral administration of probiotic Lactobacillus plantrum NRRL B-227 in vivo using mice was determined.2 g Magnesium sulfate heptahydrate. .
8. Meat extract. Magnesium sulfate tetrahydrate. MRS liquid medium composed of Peptone. The organic phase was concentrated to dryness under vacuum by using a rotary evaporator.0 Triammonium citrate. In the HPLC protocol.6. Diethyl ether.0. D(+) Glucose. the frequent experimentation shows that 100 A pore columns are also effective for peptides purification. In the purification protocols described by the authors listed in the references of this chapter.2. 30. 10.05 and distilled water up to 1000ml. 2.0. Gels were stained for protein using Coomassie Blue as described by .8 and 1 ml M-dithiothreitol. gels and gel buffer were prepared as described by [38. 4 (2): 115-125. sakaguchi.2 before sterilization at 30°C for 48 h . pH Resistance: purified peptide antibiotic (400 µl) were adjusted to pH 2. ehrlish. Heat Resistance: purified peptide antibiotic (400 µl) was exposed to various heat treatments: 40. This analytical technique has been shown to be extremely valuable for the analysis of this peptide antibiotic. 20.0. the peptide antibiotic extract is loaded onto the HPLC column. followed by filtration of the supernantant through a 0.0 ml/min. 4. Dialysis: was carried out against the same buffer for 18 h in spectrapor dialysis tubing. yeast extract. 2009 Fermentation.10 and 12 with hydrochloric acid and sodium hydroxide. Precipitation and Purification Fermentation: Lactobacillus plantrum NRRL B-227 was propagated when 1L MRS broth inoculated with 10 ml bacterial culture and incubation for 48 h in a static incubator at 30°C. ferric chloride and mayer reactions . The inhibitory activity is found in fractions collected in between the retention time of the peptides (hatched area). Assay of the antimicrobial activity was determined. Physico-chemical Properties Spectroscopic Analysis: The IR. 45]. Reaction of the Antibacterial Agent with Certain Chemical Test: For this purpose. The pellet was re-suspended in 25 ml of 0. n-propanol. Various organic solvents including iso–amylalcohol. 39].0. Extraction. This product was named CE1 . petroleum ether were added to purify antimicrobial substance in 1:1 ratio. Although for peptide separation a 300 ? pore column is sometimes recommended. Aliquot volumes of each fraction were then removed after 0. 100 and 120°C. 60 or 90 min . 0.05 M potassium phosphate buffer pH 7.2 um pore size cellulose acetate filter. with 10 % polyacrylamide gels as described by . 80. purifications were performed by the frequent use of C18 column (hypersil ODS) a column. Precipitation: The precipitation process of the peptide antibiotic was carried out using ammonium sulfate. at 4°C.0 Dipotassium hydrogen phosphate. 20. 8. Culture supernatant was treated with solid ammonium sulphate to 0. since peptide antibiotic are generally resistant to different organic solvents used as mobile phases and the high pressures employed trough the chromatographic process.227 was identified according to the recommended international references [43. UV and Mass spectrum were determined at the National Research Center Egypt and amino acid analyzer was determined at the Desert Research Center Egypt. Separation of Antibacterial Agent by SDS-PAGE: The pure peptide antibacterial agent was checked by sodium dodecyl sulphate-polysaccharide gel electrophoresis (SDS-PAGE). In the protocols used in our laboratory. ninhydrin.4.1 % SDS in pH 8. hexane.. Purification: The purification of the peptide antibiotic was carried out by using reverse-phase (RP) HPLC. fehling. incubated for 4 h at room temperature [35. The pH was adjusted at 6. nitroprusside. 40 and 60% saturation.1% trifluoroacetic acid (TFA). Biotech.000 rpm for 20 min. 60. The mobile phase consisted of (i) acetonitrile and (ii) HPLC-grade water containing 0.0 Magnesium sulfate heptahydrate. Extraction: A cell-free filtrate was obtained by centrifuging (10. The sample was loaded on the C18 column (hypersil ODS) and separated by a linear biphasic gradient of 20 to 80% acetonitrile over 30 min at a flow rate of 1. In the presence of 0. Sodium acetate trihydrate. 42]. 117 . the following reactions were carried out: molish’s.0. several types of RP columns bas been used. after the sample has been separated by cation exchange or SPE chromatography. 0. chloroform. 5. being the silica-based C4 to 18 ODS the most frequently employed. The mixtures were stirred for 2 h at 4°C and later centrifuged at 9. Characterization of the Antibacterial Agent: The peptide antibiotic produced by Lactobacillus plantrum NRRL B. 2.Global J. & Biochem.000 rpm for 50 min at 4°C.
The first group of animals was kept without any treatment (the control group). The group D of animals was orally treated with ximacef antibiotic (Dose 5. On the basis of the previously collected data. used sterile inoculated lob for inoculation each stool sample on three selective plates media S.Global J.S. Group of animals from B to D were orally challenged twice after disinfection of cages by ethyl alcohol 70 % with 5 µl per mouse of saline (NaCl 0. Treatment Step: The group B of animals did not receive any antibiotic and lyophilize culture of L. while chloroform extraction completely destroyed the antibacterial substance activity. agar. The group F of animals was challenged twice orally with lyophilized culture of Lactobacillus plantarum with dose of 0.5 g per mouse once/ day) for 5 days with disinfected cages by ethyl alcohol 70 % every morning. at 4°C. NRRL B-227 may be belonging to Lactobacillus plantrum NRRL B-227. The C group of animals was treated with lyophilized culture of L. However. while grew luxuriously at 37°C and pH of 9.227 (Dose= 0. antibacterial substance was removed from the aqueous phase and recovered from the organic phase (Table 2).5 g per mouse once day) and Ximacef antibiotic (Dose= 5.0.227 only. plantarum NRRL B. in each case.2 um pore size cellulose acetate filter. Fermentation of Antibacterial Agent: Lactobacillus plantrum NRRL B-227 was propagated when 1L MRS broth inoculated with 10 ml bacterial culture and incubation for 48 h in a static incubator at 30°C. & Biochem. after 5 days stool samples were taken for analysis. followed by filtration of the supernatant through a 0.000 rpm for 20 min. Biotech. di-ethyl ether and petroleum ether did not result in the removal antibacterial substance produced at the aqueous phase to the organic phase. 1). MacConkey agar and Blood agar under aseptic condition. RESULTS Screening for the Antibacterial Activities: The active metabolites produced by lactobacillus sp NRRL B-227 118 exhibited various degrees of activities against Gram positive and Gram-negative target strains.227.4. whereas Catalase and nitrate reduction are negative. arabinose and starch. the reference sequence was retrieved from the Gene Bank databases.9 %) suspension mixture (Staphylococcus aureus ATCC 29213. each group containing 3 mice. The bacterial strain fermented mannitol. when different alcohols such as n-propanol and Iso-amyl alcohol were used in the extraction procedure. The group E of animals was treated orally with lyophilized culture of L. rhamnose. It was observed that extraction with polar solvents such as hexane. 4 (2): 115-125. A cell-free filtrate was obtained by centrifuging (10. 2009 Application of Probiotic Lactobacillus plantrum NRRL B-227 In Vivo Using Mice Animals: A total number of 18 male Egyptian mice weighing approximately 20 g were kept in separate cylindrical wire cages and were supplied with pellet food and drinking water. plantarum NRRL B. Inoculation in Mice: The animals were divided into six groups.2 g / kg b. Extraction of Antibacterial Substance with Organic Solvents: Various organic solvents were tested for the extraction of peptide antibiotic produced by the L. raffinose and trehalose but they could not ferment xylose. ribose.2 gm / kg b.wt per mice once day) for 5 days with disinfected cages by ethyl alcohol 70 % every morning. Bacterial Identification: The bacterial strain.227. especially human intestinal bacterial pathogens (Table 1). almost compete sequence of type strains of family lactobacilli and then with corresponding sequences of representative Lactobacillus sepecies. melibiose. galactose. maltose. Their growth was weak pH of 4. sucrose. The bacterial strain produced lactic acid and degradable of esculine. . glucose. lactose. plantarum NRRL B. plantarum NRRL B. were found to be Gram-positive bacilli and hydrolysis gelatine. respectively orally (Dose=0.5 g per mice mouse once / day for 5 days. incubated at 37°C for 24 h then the plates were examined to determine the bacterial count. Escherichia coli ATCC 25922 and Salmonella typhi).. The phylogenetic tree (diagram) revealed that the bacterial strain is closely related to Lactobacillus plantrum with similarity matrix of 100% (Fig.wt per mice once day) for 5 days with disinfected cages by ethyl alcohol 70% every morning. Sensitive balance was used for weighting mice ever day. Molecular Phylogeny: The resulted sequence was aligned with available. mannose. Stool Culture and Body Weight: A stool sample was collected early from each mice cage in sterile container. it could be stated that bacterial strain.
During the purification procedure. 1650. The phylogenetic tree was based on the pairwise comparisons of 16S DNA sequences.0 0. plantarum NRRL B. collinoides Lacto. pentosus Lacto. 199.0 22.Propanol Hexane Di-ethylether Petroleum ether *The diameter of the used cup assay was 9 mm 100% 95% 90% Lactobacillus sp Lacto.0 21. Assay of the antibacterial activity was carried out increase of antibacterial activity comparing with culture supernatant.0 10.0 22. plantarum Lacto.05 M potassium phosphate buffer pH 7. The Mass spectrum showed that the molecular weight at 381 (Fig.0 22.5). Dialysis was carried out against the same buffer for 18 h in spectrapor dialysis tubing.0 20. The mixtures were stirred for 2 h at 4°C and later centrifuged at 10. The antibacterial peptide exhibited *The diameter of the used cup assay was 9 mm Table 2: Extraction of antibacterial substance by organic solvents *Mean diameter of inhibition zones (mm) -------------------------------------------------------------Organic phase Aqueous phase 11. paracollinoi Lacto. Precipitation of Peptide Antibiotic by Ammonium Sulfate: The peptide antibiotic produced by L.0 Organic solvent Iso-amylalchohal Chloroform n. The pellet was resuspended in 25 ml of 0.71 cmG1 (Fig.7.0 22.0 21. each step resulted in a considerable loss of protein concentration while activities are increases. & Biochem. The amino acid composition of the peptide antibiotic obtained after the final reverse-phase chromatography was determined. Biochemical Reaction of the Peptide Antibiotic: The reactions revealed the detection of certain groups in the investigated molecule. The active fraction was purified by a subsequent reverse phase chromatography and its amino acid sequence was determined (Fig. The fractions have been given from Reverse-phase (RP) HPLC are assayed to determined the antibacterial activity then taken the active fraction and estimated the total protein concentration.12.0 22.000 rpm for 50 min at 4°C.227 was treated with ammonium sulphate 60% 119 .227.51. 285.0 23.0 0. Purification of Peptide Antibiotic by Reverse-phase (RP) HPLC: Partial purification steps of the peptide antibiotic are summarized in Table 4.0 Test organism Staphylococcus aureus Micrococcus luteus Bacillus pumilus Bacillus subtilis Bacillus subtilis Escherichia coli Escherichia coli Salmonella typhi Enterobacter cloacae Pseudomonas aeruginosa Klebsiella pneumonia Klebsiella pneumonia saturation. brevis Lacto.7and 3431. Separation Peptide Antibiotic by Polyacrylamide Gel Electrophoresis: The active proteins were homogenous in disc polyacrylamide gel electrophoresis and gave only one band of protein at molecular weight 3. 3).0 0. 4 (2): 115-125.8 KDa.0 20. 2009 Table 1: Mean diameters of inhibition zones (mm) caused by 100µl of the antibacterial activities produced by Lactobacillus sp NRRL B227 in the agar plate diffusion assay.77.0.. Biotech.0 0. 4). On the other hand the estimation of total protein by lowery method was carried out decrease of total protein content (Table 3).0 0. 2). 1400. plantarum NRRL B.0. arizonensis Lacto.Global J.0 11. Origin ATCC 29213 ATCC 9341 NCTC 8214 NRRL B-543 NCTC 10400 ATCC 25922 NCTC 10416 NCIMB 9331 ATCC 13047 ATCC 10145 NCTC 9111 ATCC 13883 *Inhibition zone (mm) 21. curvatus 100% 100% 100% 99% 94% 94% 99% 92% 90% Fig. strain among neighboring species. Amino Acid Composition of Peptide Antibiotic The Amino Acid Content Was Carried out Using: Amino acid analyzer LC3000.0 0.0 0. maximal IR spectra showed eight absorption peaks in the region of 1129. 5.0 19. The ultraviolet (UV) absorption spectrum are recorded a maximum absorption peak at 328 and 336 nm (Fig. 2919.0 13.. fornicalis Lacto.0 0. Spectroscopic Characteristics: The spectroscopic analysis of the purified of peptide antibiotic produced by L. 1: The phylogenetic position of the Lactobacillus sp. The calculation of the number of amino acid residues in the antibacterial substance revealed that it contained 13 amino acids (Table 4). rapi Lacto.
R spectrum of peptide antibiotic produced by Lactobacillus plantrum NRRL B-227 Fig.. 2: Purification of peptide antibiotic produced by Lactobacillus plantrum NRRL B-227 using HPLC Fig. Biotech. 4 (2): 115-125.Global J. 4: Ultraviolet absorbance peptide antibiotic produced by Lactobacillus plantrum NRRL B-227 120 . 2009 Fig. & Biochem. 3: I.
ehrlish and nitroprusside reactions.808896 ND 7.318516 2. plantarum NRRL B.23 -65. fehling.25 48. sakaguchi.790077 ND 0.65333 ND 1.227 No 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 R.822643 5. Antibacterial agent produced by Lactobacillus plantrum NRRL B-227 activity remained constant after heating at 121°C for 10 min. it was found to be stable at pH 4 to 9.399466 0.77 68.70 25. 2009 Fig.59 -24. Identification of the Peptide Antibiotic: On the basis of the recommended keys for the identification of antibiotics and in view of the comparative study of the recorded properties of the peptide antibiotic. Application of Probiotic Lactobacillus plantrum NRRL B-227 in Vivo Using Mice's: In the present studies.Global J..291149 1. whereas negative results with molish’s.22 Amino acids Aspartic acid Therionine Serine Glutamic acid Proline Glycine Alanine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Histidine Lysine Ammonia Argenine % 0.20 56. Recovery percentage is the remaining protein concentration as a percentage of the initial protein concentration Table 4: The amino acid composition of the peptide antibiotic produced by L. Heat Resistance: The effects of heat on peptide antibiotic were determined by using Escherichia coli. it could be stated that the antibacterial agent is suggestive of being belonging Bacteriocin antibiotic.0 IZ (mm)a 17. Protein concentration was determined by the lowery method c.294045 as indicator organism.0 35 50 Protein (mg\ml)b 4.162441 0. 5: Mass spectrum of peptide antibiotic produced by Lactobacillus plantrum NRRL B-227 Table 3: Precipitation and purification of peptide antibiotic produced by Lactobacillus plantrum NRRL B-227 Purification stages Culture supernatant Amm.87712 0.(CE1) HPLC Volume (ml) 1000 20 1.19 -40.95 17. Inhibition zone including the diameter of the well 9 mm b.57 15.77 -36.T 11.44 19.6 2. ATCC 25922 as indicator organism.13 Recovery (%)c 100 55 3.46419 1. 4 (2): 115-125.1 a.651119 ND 76. Antibacterial agent produced by Lactobacillus plantrum NRRL B-227. & Biochem. pH Resistance: The effects of pH on peptide antibiotic were determined by using Escherichia coli. Biotech.59 44. The group C are infected with pathogenic bacteria could be recorded recovery healthy when treatment with probiotic Lactobacillus plantarum NRRL B-227 after 5 days and total mice's weight in group F that increase of body weight from 20 gm to 26 gm when consumed probiotic Lactobacillus plantarum NRRL 121 positive results with ninhydrin. ferric chloride and mayer reactions. ATCC 25922 .SO4 ppt.46701 0.5 0. orally administered lactobacilli strains did flourish in the intestinal tract because they could overcome host defense mechanisms.
almost compete sequence of type strains of family lactobacilli and then with corresponding sequences of representative Lactobacillus species. Forty lactobacillus sp were selected from American agriculture culture collection. aureus growth Count -------------------------------------------------------------------------------------------------------------------------------Groups A Control Binfected without treatment C Infected and treatment with L.. sucrose. plantrum D Infected and treatment with antibiotic E Infected and treatment with both L. the broad spectrum strain Lactobacillus sp NRRL B-227 was exhibited various degrees of activities against bacterial pathogens. Enterobacter cloacae ATCC 13047.0. Among the forty Lactobacillus sp. Gram positive bacteria. maltose.----------------------------------------------------------E. in each case. & Biochem. The resulted sequence was aligned with available. 23. Klebsiella pneumonia NCTC 9111.05 M potassium phosphate buffer pH 7. The phylogenetic tree (diagram) revealed that the bacterial strain is closely related to L. coli growth Count S. Biotech. Their growth was weak at pH of 4. typhi growth Count S. plantrum with similarity matrix of 100%. whereas the group B and D are death after 5 days for treatment when infected by bacterial pathogenic (Table 5). typhi growth Count S. 48]. The active metabolite of the peptide antibiotic was precipitated by ammonium sulphate (60% saturation). raffinose and trehalose. they luxuriously grew at 37°C and pH of 9. plantrum and antibiotic F Treatment with L. The pellet was resuspended in 25 ml of 0. Staphylococcus aureus ATCC 29213. The bacterial strain was fermented mannitol. All these strains were screened for their antibacterial activity against pathogenic bacteria. Pseudomonas aeruginosa ATCC 10145. Klebsiella pneumonia ATCC 13883 [47. rhamnose. lactose. Salmonella typhi NCIMB 9331. mannose. galactose. The fractions have been assayed to determined the antibacterial activities then taken the active fraction and estimated the total protein concentration . while.Global J. Bacillus subtilis NRRL B-543. Bacillus subtilis NCTC 10400 and Gram negative bacteria. arabinose and starch [49. The mixtures were stirred for 2 h at 4°C and later centrifuged at 10.. melibiose. The amino acid .000 rpm for 50 min at 4°C. Escherichia coli NCTC 10416. plantrum 50000 No growth 30000 No growth 100000 No growth Death No growth Death No growth Death No growth 100000 25000 100000 Death Death Death 25000 25000 100000 No growth No growth No growth Recover healthy Death of all individual Death of all individual Healthy and Increase of body weight from 20gm to 26 gm 25000 25000 50000 Death Death Death No growth No growth Report of stool culture after 5 days No growth No growth No growth No growth Comment Healthy control Death of all individual B-227 and significantly from the control. ribose.4. 51]. 4 (2): 115-125. The purification was performed using reverse-phase high performance liquid chromatography (RP-HPLC) on ODS Hypersil C18 column . glucose. whereas catalase and nitrate 122 reduction are negative. The bacterial strains were found to be Gram-positive bacilli and hydrolysis gelatine. but they could not ferment xylose. aureus growth Count E. especially human intestinal bacterial pathogens. coli growth Count S. Escherichia coli ATCC 25922. Bacillus pumilus NCTC 8214.0 [22. the reference sequence was retrieved from the Gene Bank databases. 2009 Table 5: The application of oral administration of probiotic Lactobacillus plantrum NRRL B-227 in vivo using mice's Treatment with Lactobacillus plantrum NRRL Infection with pathogenic bacteria B-227 and/or ximacef antibiotic -------------------------------------------------------------. The strain produced lactic acid and degradable of esculine. DISCUSSION Probiotics were reported as a food supplement and improvement for human health through production of useful compounds . Micrococcus luteus ATCC 9341. 50].
M.. The reactions revealed the detection of certain groups in the investigated molecule.8 KDa. plantarum NRRL B. 1989. A. G. 1994.. The group C are infected with pathogenic bacteria could be recorded recovery healthy when treatment with probiotic Lactobacillus plantarum NRRL B-227 after 5 days and total mice's weight in group F that increase of body weight from 20 gm to 26 gm when consumed probiotic Lactobacillus plantarum NRRL B227 and significantly from the control. 78(78): 349-58.. London: Blackie Academic and Professional. Control of Listeria monocytogenes in ground beef by Lactocin 705. Fadda. Holgado and G. fehling. sliced vacuum. 27(27): 397-402.. Microbiology.N. 2. whereas the group B and D are death after 5 days for treatment when infected by bacterial pathogenic . 123 . The ultraviolet (UV) absorption spectrum are recorded a maximum absorption peak at 328 and 336 nm. sliced vacuum. 2919. sakaguchi. Arca. Control of Listeria monocytogenes in ground beef by Lactocin 705. 1. Vignolo. Rekhif. Atrih. B. 42: 119-126.J. Lindgren. it could be stated that the peptide antibiotic is suggestive of being belonging Bacteriocin antibiotic [15. 5. S. A. 1650. Orally administered lactobacilli strains did flourish in the intestinal tract because they could overcome host defense mechanisms. and W. P. Activity of plantaricin SA6. Vignolo. Food Technol.E. 1996. Detection.0. ferric chloride and mayer reactions. Holck. 3. & Biochem. Int.. Appl Environ Microbiol. S. J. 1994..C. 8.51. A. a bacteriocin produced by Lactobacillus casel CRL 705. a bacteriocin produced by Lactobacillus plantarum SA6 isolated from fermented sausages. De-Ruiz. and D. De Martinis. ATCC 25922 as indicator organism was exhibited the activity remained constant after heating at 121°C for 10 min and its stable at pH 4 to 8 respectively. Food Microbiol. S. Health and nutritional benefits from lactic acid bacteria. Mayo and J. 5.and gas-packaged meat. T. T. The spectroscopic analysis of the purified of antibacterial compound produced by L. 11. Probiotics had been used as growth promoters due to their ability to suppress the growth and activities of growth depressing microflora and their ability in enhancing absorption of nutrients through the production of digestive enzymes Chang. Bredholt. Vandamme. 199. 2009 composition of the peptide antibiotic revealed that it contained 13 amino acids . ISBN 0-75140174-9.. J. M. 53(53): 43-52. 6: 2158-63. N. Oliver. S. Int. Holck. E. Holgado and G. 87(87): 149-64.A. S. 4. 1996..71 cmG1. Food Microbiol. The identification of peptide antibiotic and in view of the comparative study of the recorded properties of the antibacterial peptide. Dobrogosz. Genetics and Applications. Bredholt. Protective cultures inhibit growth of Listeria monocytogenes and Escherichia coli 0157:H7 in cooked.77. Int.. Michel and G. 87: 175-8. Lefebvre.and gaspackaged meat. purification and partial characterization of plantaricin C.M.12.G. Nesbakken and A. Food Microbial. a bacteriocin produced by a Lactobacillus plantarum strain of dairy origin. Inhibition of Listeria monocytagenes in a ponk product by a Lactobacillus sakei strain. Biotech. 43(43): 164-9. It could be concluded that the bacteriocin antibiotic produced by probiotic Lactobacillus plantarum NRRL B-227 that demonstrated inhibitory affects against bacterial pathogenic.. FEMS Microbiol Rev. Suarez. The peptide antibiotic exhibited positive results with ninhydrin.P. Gonzales. De Kairuz. Int J Food Microbiol.Global J. Antagonistic activities of lactic acid bacteria in food and feed fermentations. Fadda. 27: 397-402. J. B. 1999. 44. 53: 43-52... Protective cultures inhibit growth of Listeria monocytogenes and Escherichia coli 0157:H7 in cooked..7 and 3431. De Vugst.227.. De Ruiz. 7. 1998. FEMS Microbiol Rev.N. J. 4 (2): 115-125. Proteins were localized by staining with coomassie blue. Bacteriocins of Lactic Acid Bacteria. 9. 1990. A. P. Nesbakken and A.J.P. The Mass spectrum showed that the molecular weight at 381. 10. a bacteriocin produced by L casel CRL 705. A pure protein was subjected to polyacrylamide gel electrophoresis (10%) in the presence of sodium dodecyl sulphate (SDS). maximal IR spectra showed eight absorption peaks in the region of 1129. De Kairuz. 45].. Franco.. Antimicrobial substances from lactic acid bacteria for use as food preservatives. J Appl Bacteriol. The active proteins were homogenous in disc gel electrophoresis and gave only one band of protein could be recorded at molecular weight 3. and E. 1995. L.. Daeschel. whereas negative results with molish’s. M. M. 1990. REFERENCES Gilliand.. ehrlish and nitroprusside reactions . A. G. 6. 1400.. Int.W. 1999. The effects of heat and pH resistances on antibacterial agent were determined by using Escherichia coli. Oliver.7. Food Microbiol. S. 285. 43.
1988. Chem.C.Global J. Microbiol.. 16. 33: 313-319.N. 27. Edition Cambridge. Aattouri. Klaenhamner. Minekus. Poultry Sci. Structural expression and evolution of a gene encoding the precursor nisin. & Biochem. 12: 247-285. Reddy. 1993. production and partial purification of Bulgaricus cultured. H. 2.. October. M. Antimicrobial activity of Lactobacillus sake isolated from meat.N.. Br.W. 82: 640-647. Plantaricins S and T. 4 (2): 115-125. M.C. Association of a 13. 1994. V. pp: 193-199. J. R. Appl. A.. Desmazeaud. Food. Riol. 84: 97-102. 2000. Appl. Hansen.. J. M.J. 24.J. A medium for the cultivation of Lactobacilli.M.. R. Lucke. S. 21. Cultural conditions for the production of bacteriocin by a native isolate of Lactobacillus delbruecki SSp. 1951. Randall. 1985. Antimicrobial activity of lactobacilli: preliminary characterization and optimization of production of acidocin B.. Kim and W. De Man. Nettles. 13... J. Dobrogosz. Sharpe. J. a small-protein antibiotic produced by Lactococcus lactis.. 18. 124 . Press. D. Hansen. Bacteriol. Microbial Ecology of Health Diseases. and F. 1993. J. T. Tome.. Casas. Farr and R. I. Some chemical and physical properties of nisin. Banerjee and J.D. 2001. M.. Marcos and D. Friend and R. 2: 132-134.6 megadalton plasmid in Pediococcus pentosaceus with bacteriocin acidity. Sci. J. Appl. J. Dairy Products.C. Aly. Chandan. 29. Biochemical and genetic characteristics of bacteriocins of food-associated lactic acid bacteria. Balasubramanyam. Environ. Ruiz-Barba. Vander Vossen. Ecol. C. Molecular microbial analysis of bifi dobacterium isolates from different environments by the species-specifi c amplifi ed ribosomal DNA restriction analysis (ARDRA). 1993. R. and S. 56: 338-356. FEMS Microbiol. Assessment of probiotic properties of a strain of Lactobacillus plantarum isolated from fermenting corn slurry. 2001. 26.T. R. H. Buchman. F. B.. 55: 1901-1906. Lowery. O. Ten Brink.D.V. Cowan.A. Antonie van Leeuwenhoek... Kim. and W.. 31. J. Appl. J.B. 52: 411-415. Osho. Oral ingestion of lactic acid bacteria by rats increases lymphocyte proliferation and interferon ã production. Biotech. 59(5): 1416-1424. 17. 36. Acad. Delves-Broughton. Fuller. 1972. 2009 12. W. J. Reniero and R. Kim. Protein measurement with Folin Phenol reagent. Natural antibiotic activity of Lactobacillus acidophilus and bulgaricus. 1960. Huis. Characterization of a Bacteriocin-Like Inhibitory Substance Produced by Lactobacillus plantarum Isolated from Egyptian Home-Made Yogurt. Bouras. 15. Chem. 193: 265-275. 1987. 1990. Lemonnier.L. Two New bacteriocins produced by Lactobacillus plantarum LPC 10 isolated from a green olive fermentation. 32 (Supplement222).H. Cowan and Steel. a novel bacteriocin produced by L. N... Environ. J. FAO/WHO. Modification of the intestinal flora using probiotics and prebioticss. Vol. 14. 2007. and M. New York..L.A. Genetics of bacteriocins produced by lactic acid bacteria. 35. Agri.L. acidophilus M46. Gastroenterology. pp: 28-31. Kavanagh. J. and J. W. 30. Asia. Elli. 50: 1538-1541.M. s Manual For The Identification Of Medical Bacteria 2nd. Environ. bulgaricus CFR 2028 in milk medium. 2001. Jimenez-Diaz. Rios-sanchez. Abdel-Bar N.R. Rosenbrough. Zink. N. 28. N.. Rill. 12: 39-85.J. M.C. Park. 22. Schillinger. 77: 140-148. A. Microbiol.. 1989. 2001. Varadaraj. S. J. Ventura. Appl. Antimicrobial Peptides and Bacteriophages. 1990. and G. 19. Microbiol. J. Daeschel.A. B. Biol.R. 2001. FEMS Microbiol. R. 32. 36: 113-121. Chang.C. K. G. Appl. 2003. 23. Nisin and its use as food preservative. Bacteriol. Food Prot. Report of a joint FAO/WHO expert consultation on evaluation of health and nutritional properties of probiotics in food including powder milk with live lactic acid bacteria (LAB). 56: 2551-2558. Univ.. G.. 33..K.F...G. E... and B. J. Y. R. Press. 44: 100-117. Joerger1.A. Alternatives to Antibiotics: Bacteriocins. Scandinavian J. Rogosa and E. Piard. 263: 16260-16266.O. 1997. Klaenhaemmer.. Microbiol. 87: 367-373.J. 8: 15-19. Food Technol. J. Selection of potential probiotic lactobacillus strain and subsequent in vivo studies. 1984.M. M.. Leer and J. Harris and R.. a small protein antibiotic. Analytical Microbiology. M. Food Sci. 2004. Barefoot. and T.J. Rev.. 25. B. R. Liu. 34. Gibson. Kim. Purification and properties of an antimicrobial substance produced by Lactobacillus bulgaricus. 20. Oyetayo. 1998. Validation of the probiotic concept: Lactobacillus reuteri confers broad spectrum protection against disease in humans and animals. 23: 130-135. Microbiol. V.H.. Shahani. 1974. Environ. Appl. Environ..
Atta. Salvucci. 53.. 35:157-160. Clin. 40.D thesis. Purification and Characterization of Bacteriocin Produced by Isolated Strain of Lactococcus lactis. H. Reultericin 6. 10: 2606-2617. H. Weiss. Tannock. 2008. Al-Azhar University. S... J.. Itoh. 1984. Vera Pingitore. 4(11): 1315-1321. Genus Lactobacillus. PrzondoMordarska. 2009 37. New York: Academy Press.A. Vijai. Lactobacillus acidophilus 30SC. Abd El-Sabour and Y. Sneath... H. 42: 179-183. 1990.L. Gibson.Global J. W..L. Baltimore: Williams and Wilkins Co. 53(2): 133-42. S..K.A. Electrophoetic analysis of the major polypeptides of the human erythrocyte membrane.T.J. Rammelsberg... Cairo. Clostridium difficile). 55. 43. a new bacteriocin produced by lactobacillus reuteri LA6. Med Doew Mikrobiol. Hansen. 2007.M.. Wallach. Applied Sci. 1990. 2005. K. 83: 2747-2752.H. J. Immunol. Holt (Eds). Journal Of Culture Collections. 47. Appl. P.. 1986. and G. 49. Sharpe. Food Chem and Biotech. The effect of dietary and environmental stress on the gastrointestinal microflora In Health and Disease.J. Samant. Campylobacter jejuni. S. 52.W. M. 54. 1990. Radler. 1997. Biotech. 2001.H.. 32 (Supplement 222): 28-31. In: Bergey’s manual of systematic bacteriology. Dairy Sci.H. ed. Some chemical and physical properties of nisin.A.A. J. G. Sesma and M. Ph. G.. 104: 237-255. Appl. Yoshihoka and T. 1971. 42. Dicks and M. Lett.. FEMS Microbiol. Exp.. Kruisselbrink A. Health and nutritional benefits from lactic acid bacteria. Vaz-Velho and P.T. J.N. Rev. Microbiol. Toba. 46. M. and J. Numerical Taxonomy Program. Compylobacter coli. Janssen. 4 (2): 115-125. P. Appl. 56: 2551-2558.. Bacterial Identification Program. H. F. Scandinavian J.. Brazilian Journal of Microbiol. Chikindas. Modification of the intestinal flora using probiotics and prebioticss. Black-Shear. Comparison Of Two Methods For Purification Of Plantaricin St31. Mair. C. O. E.S. 48. N. Kadirvelu. Ibrahim.J. Appl. 2001. M. Environ. 1987. Biochemistry. Hentges. Kim and R. 51. Gosciniak and A. J. C. J. 38. P..... E.E.E. 87: 175-178. Characterization and Purification of a Bacteriocin Produced by a Potential Probiotic Culture. Marilingappa and J. E. a small-protein antibiotic produced by lactococcus lactis. Gastroenterology. 1999. Steck and D. Fuller. 39. Different strategies for purification of antimicrobial peptides from Lactic Acid Bacteria (LAB). Anatgonistic activity of Lactobacillus bacteria strains against anaerobic gastrointestinal tract pathogens (Helicobacter pylori. Svetoslav.E. purification and partial characterization of plantaricin 423. Bacteriol. Communicating Current Research and Educational Topics and Trends in Applied Microbiol. 84: 1131-1137. A Bacteriocin Produced By Lactobacillus Plantarum St31. R.E. pp: 517-539. Microbiol. Faculty of Science. 1983. Nader-Macías. 2: 1209-1234.. Microbiol. Mostafa.F. S. T. El-Shafie. Systems for polyacrylamide gel electrophoresis" Methods Enzymol. Valtonen. Isolation. Egypt.W.R. M. 125 . Heijne Den Bak-Glashouwer. J. Strus. 44. M. Thole and R. Van Reenen. Oh. Kandler. 45. Application of biotechnology in search for antibiotics from environmental polutents under solid state fermentation conditions. Gibbs. L. Pakosz.. 2000. Res. and N. 1998. N. Isolation And Characterization Of Bacteriocin Producing Lactic Acid Bacteria From A South Indian Special Dosa (Appam) Batter. D. pp: 557-568. 41... Recombinant Lactobacillus plantarum inhibits house dust mite-specific T-cell responses. Fairbanks.M. a bacteriocin produced by Lactobacillus plantarum.Lactobacillus species Belgian J. 50..G. & Biochem. 126: 2-8. 2004. 13: 281-286. and F. 4: 53-60. Worobo.E. Antibacterial polypeptides of Lactobacillus species. D. Havenith. 1991. T. Liu. 69: 177-184.
This action might not be possible to undo. Are you sure you want to continue?