Purification and Characterization of Plantaricin ZJ316, a Novel Bacteriocin against Listeria monocytogenes from Lactobacillus plantarum ZJ316

2018 ◽  
Vol 81 (12) ◽  
pp. 1929-1935 ◽  
Author(s):  
LIN CHEN ◽  
QING GU ◽  
PING LI ◽  
YANJUN LI ◽  
DAFENG SONG ◽  
...  

ABSTRACT Bacteriocins are known to be natural preservatives, which are becoming increasingly necessary in many types of food to control the proliferation of pathogenic bacteria. In this study, a novel bacteriocin produced by Lactobacillus plantarum ZJ316, called plantaricin ZJ316, was purified by ammonium sulfate precipitation, gel chromatography, and high-performance liquid chromatography. By mass spectrometry, the molecular mass of plantaricin ZJ316 was determined to be 2,366.06 Da. No homologous sequences were found in databases based on comparisons with the N-terminal amino acid sequencing. The bacteriocin was heat resistant and stable after incubation at pH 2.0 to 10.0. It was sensitive to α-chymotrypsin, trypsin, and proteinase K. Plantaricin ZJ316 had a broad inhibitory activity against gram-negative and gram-positive bacteria, especially Listeria monocytogenes. Our results suggested that this bacteriocin has the potential to inhibit pathogenic bacteria in food products.

2011 ◽  
Vol 76 (5) ◽  
pp. 699-707 ◽  
Author(s):  
Svetlana Seatovic ◽  
Jelena Jovanovic-Novakovic ◽  
Gordana Zavisic ◽  
Zeljka Radulovic ◽  
Marija Gavrovic-Jankulovic ◽  
...  

The aim of this study was the partial characterization of the antimicrobial peptide bacteriocin G2 produced by probiotic bacteria Lactobacillus plantarum G2, which was isolated from a clinical sample of a healthy person. Antimicrobial substance was secreted in the supernatant of an L. plantarum G2 culture, and showed a diverse spectrum of antimicrobial activity of all the tested strains of the genera Lactobacillus and the pathogenic bacteria Staphylococcus aureus and Salmonella ?bony. Isoelectric focusing revealed that bacteriocin G2 is a cationic peptide (pI about 10) with a molecular mass of 2.2 kDa according to tricine-sodium dodecyl sulphate-polyacrylamide gel electrophoresis, SDS-PAGE. The antimicrobial activity of bacteriocin G2 was diminished by the proteolytic action of trypsin and proteinase K. Bacteriocin G2 preserved its biological activity in the temperature range 40-60?C (15 min), which was lost at 80?C. Bacteriocin G2 was stable in the pH range 2-9, while treatment with 1 % Tween 80 and 1 % urea resulted in increased antimicrobial activity. The probiotic strain L. plantarum G2 produces the antimicrobial substance proteinaceous in nature with bacteriocin characteristics. Bacteriocin production is one of the key properties of probiotic bacteria with clinical potential as anti-infective agents, which will increase the likelihood of its in vivo efficacy.


2021 ◽  
Author(s):  
Amrutha Bindu ◽  
Lakshmi Devi

Abstract The focus of present study was to characterize antimicrobial peptide produced by probiotic cultures, Enterococcus durans DB-1aa (MCC4243), Lactobacillus plantarum Cu2-PM7 (MCC4246) and Lactobacillus fermentum Cu3-PM8 (MCC4233) against Staphylococus aureus and E. coli. The growth kinetic assay revealed 24 h of incubation to be optimum for bacteriocin production. The partially purified compound after ion-exchange chromatography was found to be thermoresistant and stable under wide range of pH. The compound was sensitive to proteinase-K, but resistant to trypsin, a-amylase and lipase. The apparent molecular weight of bacteriocin from MCC4243 and MCC4246 was found to be 3.5 KDa. Translated partial amino acid sequence of plnA gene in MCC4246 displayed 48 amino acid sequences showing 100% similarity with plantaricin A of Lactobacillus plantarum (WP_0036419). The sequence revealed 7 β sheets, 6 α sheets, 6 predicted coils and 9 predicted turns. The functions on cytoplasm show 10.82 isoelectric point and 48.6% hydrophobicity. The molecular approach of using Geneious Prime software and protein prediction data base for characterization of bacteriocin is novel and predicts “KSSAYSLQMGATAIKQVKKLFKKWGW” as peptide responsible for antimicrobial activity. The study provides information about broad spectrum bacteriocin in native probiotic culture and paves a way towards its application in functional foods as biopreservative agents.


2006 ◽  
Vol 69 (8) ◽  
pp. 1937-1943 ◽  
Author(s):  
PONGSAK RATTANACHAIKUNSOPON ◽  
PARICHAT PHUMKHACHORN

Lactobacillus plantarum N014 was isolated from nham, a traditional Thai fermented pork, and exhibited antimicrobial activity against Listeria monocytogenes. Its bacteriocin had a broad inhibitory spectrum toward both gram-positive and gram-negative bacteria. The bacteriocin activity was sensitive to all proteolytic enzymes used in this study, including papain, pepsin, pronase E, proteinase K, and trypsin, but was resistant to the other enzymes, such as α-amylase, lipase A, and lysozyme. Furthermore, activity was stable over various heat treatments and pH values. The bacteriocin exerted a bacteriolytic mode of action. It was produced during the exponential growth phase and reached its highest level as producer cells entered the stationary phase. Adsorption of the bacteriocin onto producer cells was pH-dependent. No bacteriocin adsorption was detected at pH 1 to 3, whereas 100% bacteriocin adsorption was found at pH 7. Plasmid isolation revealed that L. plantarum N014 contained no plasmids. From Tricine–sodium dodecyl sulfate–polyacrylamide gel electrophoresis and growth inhibition testing against L. monocytogenes, the estimated molecular mass of L. plantarum N014 bacteriocin was 8 kDa.


2015 ◽  
Vol 11 (2) ◽  
Author(s):  
N. Nofisulastri ◽  
Zaenal Bachruddin ◽  
Eni Harmayani

objectives were to study the growth pattern of Pediococcus sp. NWD 015 and bacteriocin activity, extractionand characterization of bacteriocin, and to determine the effect of storage time and temperature on bacteriocinactivity. Results showed that the bacteriocin activity increased during growth and reached the highest activity duringstationary phase. The maximum bacteriocin production reached after incubation of the cell for 12 h at 37oC in TGEbroth and decreased after 96 h incubation. Extraction with adsorbtion-desorbtion method could increased a specificactivity of bacteriocin. Bacteriocin from Pediococcus sp. NWD 015 is inactivated by Proteinase-K; however it is stillactive by heat treatment at 121oC for 15 min and over pH 2 – 11. Bacteriocin of Pediococcus sp. NWD 015 was effectiveagaints Enterococcus faecalis, Staphylococcus aureus, Eschericia coli, Listeria monocytogenes but not against Salmonellathypimurium. The molecular weight of bacteriocin is 4.95 kDa.Keywords : Bacteriocins, Pediococcus sp NWD 015.


2009 ◽  
Vol 55 (No. 7) ◽  
pp. 305-310 ◽  
Author(s):  
Ma Yuan ◽  
Pu Shangrao ◽  
Cheng Qingsu ◽  
Ma Mingdong

A new, effective and economical method to extract ardicrenin from <I>Ardisia crenata</I> Sims collected in the Wolong natural reserve, Sichuan, China, is established. <I>Ardisia crenata</I> Sims powder is counter-current extracted with 80% methanol reflux, decompressively enriched and centrifuged to defat. Supernatant is applied to macroporous resin column (AB-8) with 80% methanol, ardicrenin is isolated by silica gel chromatography with dichlormethane-acetoacetate-methanol (4:1.5:1) washing, and recrystallized in methanol. The final product which proved to be ardicrenin by analytic procedure including Furier transform infrared (FTIR) and ultraviolet spectrum (UV), mass spectroscopy (MS), nuclear magnetic resonance (NMR) and high performance liquid chromatography (HPLC) is white amorphous powder with yield of 1.59 ± 0.02%.


2015 ◽  
Vol 11 (1) ◽  
pp. 51-59 ◽  
Author(s):  
Xuan Zhu ◽  
Lei Shen ◽  
Jiao Liu ◽  
Chen Zhang ◽  
Qing Gu

Abstract Plantaricin ZJ217 was continually purified by XAD 1180, cation exchange chromatography, gel chromatography, and high performance liquid chromatography (HPLC) system. The peptide functioned as bactericidal, but did not lead to lysis of cells. Considering the potassium efflux experiment, pores may be formed in the surface of cell membrane. Fifteen of twenty amino acids identified by Edman degradation indicated that it may be a novel bacteriocin as no bacteriocin shared similar sequences. This bacteriocin exhibited strong heat stability (121°C, 30 min) and pH stability (pH 2.0–6.0). It was sensitive to proteinase K, trypsin, papain, and pepsin. This bacteriocin inhibited growth of methicillin-resistant Staphylococcus aureus (MRSA) and other bacteria.


2021 ◽  
Vol 49 ◽  
Author(s):  
Juliana Sousa Bogea ◽  
Luciane Manto ◽  
Jucilene Sena Dos Santos ◽  
Lara Franco Dos Santos ◽  
Franciele Maria Gotardo ◽  
...  

Background: Listeria monocytogenes is a pathogenic bacterium that can contaminate food and cause public health problems due its ability to form biofilms and resistance to sanitizers, it is responsible for sanitary and economic losses in food producing establishments. The difficulties in controlling biofilms and increasing resistance to traditional antibacterial agents is motivating studies of alternative potential biological agents for the control of pathogenic biofilms, among which lactic acid bacteria (LABs) are included. The objective of this work was to evaluate the activity of LABs against Listeria monocytogenes biofilm formation on polystyrene plates, a surface commonly used in the food industry.Materials, Methods & Results: Lyophilized commercial strains of Bifidobacterium animalis, Lactobacillus fermentum, Lactobacillus plantarum, Lactobacillus salivaris and Lactobacillus acidophilus were used. The strain of Listeria monocytogenes (L4) was isolated from polystyrene mats from a poultry slaughterhouse cutting room and demonstrated the ability to attach to microplates and resistance to sanitizers (sodium hypochlorite and hydrogen peroxide) at all times, temperatures and tested surfaces. The antimicrobial activity of LABs was evaluated by the agar diffusion method. The LABs that presented action on Listeria monocytogenes were selected for the inhibition and/or removal of biofilms in microplates, and all experiments were carried out in triplicate. Only Bifidobacterium animalis and Lactobacillus plantarum demonstrated action against Listeria. monocytogenes in the agar diffusion assays and were selected for inhibition and competition assays. Furthermore, competition of LABs against Listeria monocytogenes adhesion was evaluated. There was no significant difference between LABs and Listeria monocytogenes, alone or in combination, at temperatures of 30ºC and 37ºC in the Listeria monocytogenes inhibition assays on polystyrene surface. The lactic acid bacteria evaluated did not demonstrate inhibition of Listeria monocytogenes adhesin testes with optical density visualization, however, it was possible to identify a reduction in Listeria monocytogenes counts with the application of Bifidobacterium animals and Lactobacillus plantarum in the testes of competition against biofilm formation. In competition tests Bifidobacterium animalis and Lactobacillus plantarum have an injunction in Listeria monocytogenes, indicating that these lactic acid bacteria can retard Listeria biofilm formation on polystyrene surfaces and thus help control the pathogen in the food industry.Discussion: A potential mechanism to control biofilm adhesion and formation of pathogens for nutrients and fixation on surfaces, multiplication factors and surfaces are a challenge in controlling biofilms of pathogenic microorganisms, alternative measures to traditional methods for inactivating pathogens and biofilm formers bacteria are necessary. In this sense, lactic acid bacteria generate high levels of bacteriocin and are effective in inhibiting the biofilm of pathogenic bacteria, however, our study did not reveal this. We verified that Bifidobacterium animalis and Lactobacillus plantarum have an inhibitory action on Listeria monocytogenes, indicating that these lactic acid bacteria can be used to delay the formation of biofilms by Listeria on polystyrene surfaces, helping to control this pathogen in food industry.Keywords: control of biofilm, pathogenic bacteria, food industry, polystyrene surface, FTDs.


PLoS ONE ◽  
2021 ◽  
Vol 16 (5) ◽  
pp. e0251514
Author(s):  
Mohamad Malik Al-adil Baharudin ◽  
Mohamad Syazwan Ngalimat ◽  
Fairolniza Mohd Shariff ◽  
Zetty Norhana Balia Yusof ◽  
Murni Karim ◽  
...  

Infections caused by methicillin-resistant Staphylococcus aureus (MRSA) have reached epidemic proportions globally. Therefore, there is an urgent need for a continuous supply of antibiotics to combat the problem. In this study, bacteria initially identified as species belonging to the Bacillus amyloliquefaciens operational group were re-identified based on the housekeeping gene, gyrB. Cell-free supernatants (CFS) from the strains were used for antimicrobial tests using the agar well diffusion assay against MRSA and various types of pathogenic bacteria. The minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and physicochemical characteristics of the CFS were determined. Based on gyrB sequence analysis, five strains (PD9, B7, PU1, BP1 and L9) were identified as Bacillus velezensis. The CFS of all B. velezensis strains showed broad inhibitory activities against Gram-negative and -positive as well as MRSA strains. Strain PD9 against MRSA ATCC 33742 was chosen for further analysis as it showed the biggest zone of inhibition (21.0 ± 0.4 mm). The MIC and MBC values obtained were 125 μl/ml. The crude antimicrobial extract showed bactericidal activity and was stable at various temperatures (40–80°C), pH (4–12), surfactants (Tween 20, Tween 80, SDS and Triton X-100) and metal ions (MgCI2, NaCI2, ZnNO3 and CuSO4) when tested. However, the crude extract was not stable when treated with proteinase K. All these properties resembled the characteristics of peptides. The antimicrobial compound from the selected strain was purified by using solvent extraction method and silica gel column chromatography. The purified compound was subjected to High Performance Liquid Chromatography which resulted in a single peak of the anti-MRSA compound being detected. The molecular weight of the anti-MRSA compound was determined by using SDS-PAGE and zymogram. The size of the purified antimicrobial peptide was approximately ~ 5 kDa. The antimicrobial peptide produced from B. velezensis strain PD9 is a promising alternative to combat the spread of MRSA infections in the future.


2021 ◽  
Vol 12 ◽  
Author(s):  
Lei Sun ◽  
Gil Rogiers ◽  
Pascal Courtin ◽  
Marie-Pierre Chapot-Chartier ◽  
Hélène Bierne ◽  
...  

A mutant of Listeria monocytogenes ScottA with a transposon in the 5' untranslated region of the asnB gene was identified to be hypersensitive to the antimicrobial t-cinnamaldehyde. Here, we report the functional characterization of AsnB in peptidoglycan (PG) modification and intracellular infection. While AsnB of Listeria is annotated as a glutamine-dependent asparagine synthase, sequence alignment showed that this protein is closely related to a subset of homologs that catalyze the amidation of meso-diaminopimelic acid (mDAP) residues in the peptidoglycan of other bacterial species. Structural analysis of peptidoglycan from an asnB mutant, compared to that of isogenic wild-type (WT) and complemented mutant strains, confirmed that AsnB mediates mDAP amidation in L. monocytogenes. Deficiency in mDAP amidation caused several peptidoglycan- and cell surface-related phenotypes in the asnB mutant, including formation of shorter but thicker cells, susceptibility to lysozyme, loss of flagellation and motility, and a strong reduction in biofilm formation. In addition, the mutant showed reduced invasion of human epithelial JEG-3 and Caco-2 cells. Analysis by immunofluorescence microscopy revealed that asnB inactivation abrogated the proper display at the listerial surface of the invasion protein InlA, which normally gets cross-linked to mDAP via its LPXTG motif. Together, this work shows that AsnB of L. monocytogenes, like several of its homologs in related Gram-positive bacteria, mediates the amidation of mDAP residues in the peptidoglycan and, in this way, affects several cell wall and cell surface-related properties. It also for the first time implicates the amidation of peptidoglycan mDAP residues in cell wall anchoring of InlA and in bacterial virulence.


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