Antimicrobial activity of Lactiplantibacillus plantarum against shiga toxin-producing Escherichia coli.

AIMS: The aim of the present work was to characterize the Lactiplantibacillus sp. LP5 strain, isolated from pork production, and identify bacteriocin-like inhibitory substances produced by this strain. METHODS AND RESULTS: In this study, LP5 was identified by species-specific PCR and 16S rRNA sequencing. Additionally, bacterial growth kinetics, antimicrobial activity, the detection of genes related to plantaricin production, and the genetic expression of plantaricins were determined. Lactiplantibacillus sp. LP5 was identified as Lactiplantibacillus plantarum. The well-diffusion test using cell-free supernatants (CFS), neutralized CFS, CFS treated with catalase, and CFS treated with proteinase K showed that inhibitory effects on a Shiga toxin-producing Escherichia coli (STEC) strain were produced by bacteriocins. The PCR technique allowed the detection of genes encoding E/F plantaricins, as well as J/K and whole genome sequencing, and bacteriocin mining analysis allowed us to confirm the presence of these plantaricins. CONCLUSIONS: We can conclude that the inhibitory effect of L. plantarum LP5 isolated from pigs against the STEC EDL933 strain could be associated with the bacteriocins production and represents a potential use as a probiotic strain.

Structural and biosynthetic diversity of plantaricins from Lactiplantibacillus.

Lactiplantibacillus plantarum (L. plantarum) produces an antimicrobial peptide known as plantaricin. Plantaricin-producing L. plantarum is of interest for its gut-friendly nature, wide range of sugar utilization, palatability, and probiotic attributes, making it a better candidate for the food industry. Numerous strains of plantaricin-producing L. plantarum have been isolated from different ecological niches and found to follow different mechanisms for plantaricin production. The mechanism of plantaricin production is sensitive to environmental factors; therefore, any alteration in the optimum conditions can inhibit/halt bacteriocin production. To regain the lost or hidden plantaricin-producing character of the L. plantarum strains under ideal laboratory conditions, it is essential to understand the mechanism of plantaricin production. Previously, discrete information on various mechanisms of plantaricin production has been elaborated. However, based on the literature analysis, we observed that a systematic classification of plantaricins produced by L. plantarum is not explored. Hence, we aim to collect information about rapidly emerging plantaricins and distribute them among the different classes of bacteriocin, followed by classifying them based on different mechanisms of plantaricin production. This may help scaleup the bacteriocin production at industrial levels, which is otherwise challenging to achieve. This will also help the reader understand plantaricins and their mechanism of plantaricin production to a deeper extent and to characterize/reproduce the peptide where plantaricin production is a hidden character. KEY POINTS: • L. plantarum produces the antimicrobial compound plantaricin. • L. plantarum has different regulatory operons which control plantaricin production. • Based on the regulatory operon, the mechanism of plantaricin production is different.

Modulation of the Gut Microbiota by the Plantaricin-Producing Lactiplantibacillus plantarum D13, Analysed in the DSS-Induced Colitis Mouse Model.

Lactiplantibacillus plantarum D13 shows antistaphylococcal and antilisterial activity, probably due to the synthesis of a presumptive bacteriocin with antibiofilm capacity released in the cell-free supernatant (CFS), whose inhibitory effect is enhanced by cocultivation with susceptible strains. An in silico analysis of the genome of strain D13 confirmed the pln gene cluster. Genes associated with plantaricin biosynthesis, structure, transport, antimicrobial activity, and immunity of strain D13 were identified. Furthermore, the predicted homology-based 3D structures of the cyclic conformation of PlnE, PlnF, PlnJ, and PlnK revealed that PlnE and PlnK contain two helices, while PlnF and PlnJ contain one and two helices, respectively. The potential of the strain to modulate the intestinal microbiota in healthy or dextran sulphate sodium (DSS)-induced colitis mouse models was also investigated. Strain D13 decreased the disease activity index (DAI) and altered the gut microbiota of mice with DSS-induced colitis by increasing the ratio of beneficial microbial species (Allobaculum, Barnesiella) and decreasing those associated with inflammatory bowel disease (Candidatus Saccharimonas). This suggests that strain D13 helps to restore the gut microbiota after DSS-induced colitis, indicating its potential for further investigation as a probiotic strain for the prevention and treatment of colitis.

Acetate and auto-inducing peptide are independent triggers of quorum sensing in Lactobacillus plantarum.

The synthesis of plantaricin in Lactobacillus plantarum is regulated by quorum sensing. However, the nature of the extra-cytoplasmic (EC) sensing domain of the histidine kinase (PlnB1) and the ability to recognize the auto-inducing peptide PlnA1 is not known. We demonstrate the key motif Ile-Ser-Met-Leu of auto-inducing peptide PlnA1 binds to the hydrophobic region Phe-Ala-Ser-Gln-Phe of EC loop 2 of PlnB1 via hydrophobic interactions and hydrogen bonding. Moreover, we identify a new inducer, acetate, that regulates the synthesis of plantaricin by binding to a positively charged region (Arg-Arg-Tyr-Ser-His-Lys) in loop 4 of PlnB1 via electrostatic interaction. The side chain of Phe143 on loop 4 determined the specificity and affinity of PlnB1 to recognize acetate. PlnA1 activates quorum sensing in log phase growth and acetate in stationary phase to maintain the synthesis of plantaricin under conditions of reduced growth. Acetate activation of PlnB was also evident in four types of PlnB present in different Lb. plantarum strains. Finally, we proposed a model to explain the developmental regulation of plantaricin synthesis by PlnA and acetate. These results have potential applications in improving food fermentation and bacteriocin production.

Plantaricin LD1 purified from Lactobacillus plantarum LD1 inhibits biofilm formation of Enterococcus faecalis ATCC 29212 in tooth model.

Plantaricin LD1 was purified to homogeneity using activity-guided chromatography. Enterococcus faecalis ATCC 29212 was found to be sensitive to plantaricin LD1 showing 13 ± 0·21 mm zone of growth inhibition. The minimum inhibitory concentration (MIC) was found to be 50 µg ml-1 against Ent. faecalis ATCC 29212. The in vitro biofilm formation by Ent. faecalis ATCC 29212 was observed, which was completely inhibited in the presence of bacteriocin. Similarly, biofilm formation was also observed on the teeth surface showing purple colour, whereas treated-teeth were clean and indicated no biofilm formation. Further, untreated cells of Ent. faecalis ATCC 29212 were found normal and plantaricin LD1-treated cells were ruptured when seen under light microscope, suggesting killing of target cells. These findings have proven the initial leads for antimicrobial and anti-biofilm activity of plantaricin LD1 against Ent. faecalis and its possible application for the treatment of endodontic diseases.

The Antibacterial Activity Mode of Action of Plantaricin YKX against Staphylococcus aureus.

We aimed to evaluate the inhibitory effect and mechanism of plantaricin YKX on S. aureus. The mode of action of plantaricin YKX against the cells of S. aureus indicated that plantaricin YKX was able to cause the leakage of cellular content and damage the structure of the cell membranes. Additionally, plantaricin YKX was also able to inhibit the formation of S. aureus biofilms. As the concentration of plantaricin YKX reached 3/4 MIC, the percentage of biofilm formation inhibition was over 50%. Fluorescent dye labeling combined with fluorescence microscopy confirmed the results. Finally, the effect of plantaricin YKX on the AI-2/LuxS QS system was investigated. Molecular docking predicted that the binding energy of AI-2 and plantaricin YKX was -4.7 kcal/mol and the binding energy of bacteriocin and luxS protein was -183.701 kcal/mol. The expression of the luxS gene increased significantly after being cocultured with plantaricin YKX, suggesting that plantaricin YKX can affect the QS system of S. aureus.

Manganese Privation-Induced Transcriptional Upregulation of the Class IIa Bacteriocin Plantaricin 423 in Lactobacillus plantarum Strain 423.

Plantaricin 423 is produced by Lactobacillus plantarum 423 using the pla biosynthetic operon located on the 8,188-bp plasmid pPLA4. As with many class IIa bacteriocin operons, the pla operon carries biosynthetic genes (plaA, precursor peptide; plaB, immunity; plaC, accessory; and plaD, ABC transporter) but does not carry local regulatory genes. Little is known about the regulatory mechanisms involved in the expression of the apparently regulationless class IIa bacteriocins, such as plantaricin 423. In this study, phylogenetic analysis of class IIa immunity proteins indicated that at least three distinct clades exist, which were then used to subgroup the class IIa operons. It became evident that the absence of classical quorum-sensing genes on mobile bacteriocin-encoding elements is a predisposition of the subgroup that includes plantaricin 423, pediocin AcH/PA-1, divercin V41, enterocin A, leucocin-A and -B, mesentericin Y105, and sakacin G. Further analysis of the subgroup suggested that the regulation of these class IIa operons is linked to transition metal homeostasis in the host. By using a fluorescent promoter-reporter system in Lactobacillus plantarum 423, transcriptional regulation of plantaricin 423 was shown to be upregulated in response to manganese privation. IMPORTANCE Lactic acid bacteria hold huge industrial application and economic value, especially bacteriocinogenic strains, which further aids in the exclusion of specific foodborne pathogens. Since bacteriocinogenic strains are sought after, it is equally important to understand the mechanism of bacteriocin regulation. This is currently an understudied aspect of class IIa operons. Our research suggests the existence of a previously undescribed mode of class IIa bacteriocin regulation, whereby bacteriocin expression is linked to management of the producer's transition metal homeostasis. This delocalized metalloregulatory model may fundamentally affect the selection of culture conditions for bacteriocin expression and change our understanding of class IIa bacteriocin gene transfer dynamics in a given microbiome.

Polydiacetylene vesicles acting as colorimetric sensor for the detection of plantaricin LD1.

The interaction of antimicrobial peptides with membrane lipids plays a major role in numerous physiological processes. In this study, polydiacetylene (PDA) vesicles were synthesized using 10, 12-tricosadiynoic acid (TRCDA) and 1, 2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC). These vesicles were applied as artificial membrane biosensor for the detection of plantaricin LD1 purified from Lactobacillus plantarum LD1. Plantaricin LD1 (200 µg/mL) was able to interact with PDA vesicles by changing the color from blue to red with colorimetric response 30.26 ± 0.59. Nisin (200 µg/mL), used as control, also changed the color of the vesicles with CR% 50.56 ± 0.98 validating the assay. The vesicles treated with nisin and plantaricin LD1 showed increased infrared absorbance at 1411.46 and 1000-1150 cm-1 indicated the interaction of bacteriocins with phospholipids and fatty acids, respectively suggesting membrane-acting nature of these bacteriocins. Further, microscopic observation of bacteriocin-treated vesicles showed several damages indicating the interaction of bacteriocins. These findings suggest that the PDA vesicles may be used as bio-mimetic sensor for the detection of bacteriocins produced by several probiotics in food and therapeutic applications.

Maltose effective improving production and regulatory biosynthesis of plantaricin EF in Lactobacillus plantarum 163.

Plantaricin EF, a kind of natural antibacterial substance, has shown inhibitory effect on most pathogen and spoilage microorganisms, which possessed great potential in food preservation. However, the lower production of plantaricin EF has limited its large-scale production and application. In this study, the effect of maltose on plantaricin EF production and its regulation mechanism in Lactobacillus plantarum 163 were investigated. Maltose significantly improved the biomass and plantaricin EF production, which increased by 3.35 and 3.99 times comparing to the control without maltose, respectively. The maximum production of plantaricin E and F in fed-batch fermentation were 10.55 mg/L and 22.94 mg/L, respectively. Besides, qPCR results showed that maltose remarkably improved transcription of plnA, plnB, plnD, plnE, plnF, plnG1 and plnH, and heighten transcription of lamR, lamK, hpk6 and rrp6. These results provided an effective method to enhance plantaricin EF production and revealed a possible regulatory mechanism from transcriptome results that hpk6, rrp6, lamK and lamR were relative to plantaricin EF production. Genes, hpk6 and rrp6, promote transcription of plnG1, whereas lamK and lamR enhance transcription of plnA, plnB and plnD, which increased plantaricin EF production. KEYPOINTS: • Maltose was proved to be effective in promoting the biosynthesis of plantaricin EF. • Maltose promoted the transcription of biosynthesis and secretion genes of plantaricin EF. • Up-regulation of genes lamR, lamK, hpk6 and rrp6 heightened the plantaricin EF production.

Genomic analysis revealed adaptive mechanism to plant-related fermentation of Lactobacillus plantarum NCU116 and Lactobacillus spp.

Lactobacillus plantarum NCU116 is the first sequenced strain derived from traditional Chinese sauerkraut (TCS). Since NCU116 manifested outstanding probiotic effects in vitro and in vivo, it is crucial to comprehend a clear genetic background for NCU116. Functional re-annotation and comparative analysis were performed to excavate the unique and representative genes in NCU116, in order to investigate its metabolic preference and adaptive mechanism. Horizontal gene transfer (HGT) seemed to occur frequently, which endows NCU116 with a strong ability to transport carbohydrates, as a strain-specific fructose/mannose-PTS was identified, and opu and osmC coding genes were retrieved as NCU116-specific. In addition, a strain-specific type I R/M system and several prophage loci were found in NCU116, which could play vital roles in self-defense mechanism. Pathways of bacterial metabolism on plant-related substrates fermentation were then generated by reconstruction of associated pathways. Moreover, a unique potential plantaricin-producing locus with high homology to that of JDM1 was defined in the genome of NCU116, which could be very important for the preservation of fermented-food. Our results would provide critical basis for the application of NCU116 in food and pharmaceuticals industries.

Bioprospecting Antimicrobials from Lactiplantibacillus plantarum: Key Factors Underlying Its Probiotic Action.

Lactiplantibacillus plantarum (L. plantarum) is a well-studied and versatile species of lactobacilli. It is found in several niches, including human mucosal surfaces, and it is largely employed in the food industry and boasts a millenary tradition of safe use, sharing a long-lasting relationship with humans. L. plantarum is generally recognised as safe and exhibits a strong probiotic character, so that several strains are commercialised as health-promoting supplements and functional food products. For these reasons, L. plantarum represents a valuable model to gain insight into the nature and mechanisms of antimicrobials as key factors underlying the probiotic action of health-promoting microbes. Probiotic antimicrobials can inhibit the growth of pathogens in the gut ensuring the intestinal homeostasis and contributing to the host health. Furthermore, they may be attractive alternatives to conventional antibiotics, holding potential in several biomedical applications. The aim of this review is to investigate the most relevant papers published in the last ten years, bioprospecting the antimicrobial activity of characterised probiotic L. plantarum strains. Specifically, it focuses on the different chemical nature, the action spectra and the mechanisms underlying the bioactivity of their antibacterial and antiviral agents. Emerging trends in postbiotics, some in vivo applications of L. plantarum antimicrobials, including strengths and limitations of their therapeutic potential, are addressed and discussed.

In vitro and in silico approach for characterization of antimicrobial peptides from potential probiotic cultures against Staphylococcus aureus and Escherichia coli.

The focus of the present study was to characterize antimicrobial peptide produced by potential probiotic cultures of Enterococcus durans DB-1aa (MCC4243), Lactiplantibacillus plantarum Cu2-PM7 (MCC4246) and Limosilactobacillus fermentum Cu3-PM8 (MCC4233) against Staphylococus aureus MTCC 96 and Escherichia coli MTCC118. The growth kinetic assay revealed 24 h of incubation to be optimum for bacteriocin production. The partially purified compound of all the three selected cultures after ion-exchange chromatography was found to be thermoresistant and stable under a wide range of pH. The compound was sensitive to proteinase-K, but resistant to trypsin, α-amylase and lipase. Comparatively, bacteriocins from L. fermentum Cu3-PM8 and L. plantarum Cu2-PM7 showed higher stability under studied parameter, hence was taken up for further investigation. The apparent molecular weight of bacteriocin from L. fermentum MCC4233 and L. plantarum MCC4246 was found to be 3.5 kDa. Further, plantaricin gene from MCC4246 was characterized in silico. The translated partial amino acid sequence of the plnA gene in MCC4246 displayed 48 amino acids showing 100 % similarity with plantaricin A of Lactobacillus plantarum (WP_0036419). The sequence revealed 7 ß sheets, 6 α sheets, 6 predicted coils and 9 predicted turns. The predicted properties of the peptide included an isoelectric point of 10.82 and a hydrophobicity of 48.6 %. The molecular approach of using Geneious Prime software and protein prediction data base for characterization of bacteriocin is novel and predicts "KSSAYSLQMGATAIKQVKKLFKKWGW" to be a peptide responsible for antimicrobial activity. The study provides information about a broad spectrum bacteriocin in native probiotic culture and paves a way towards its application in functional foods as a biopreservative agent.

The functional capacity of plantaricin-producing Lactobacillus plantarum SF9C and S-layer-carrying Lactobacillus brevis SF9B to withstand gastrointestinal transit.

BACKGROUND: We evaluated the functional capacity of plantaricin-producing Lactobacillus plantarum SF9C and S-layer-carrying Lactobacillus brevis SF9B to withstand gastrointestinal transit and to compete among the gut microbiota in vivo. Considering the probiotic potential of Lb. brevis SF9B, this study aims to investigate the antibacterial activity of Lb. plantarum SF9C and their potential for in vivo colonisation in rats, which could be the basis for the investigation of their synergistic functionality. RESULTS: A plantaricin-encoding cluster was identified in Lb. plantarum SF9C, a strain which efficiently inhibited the growth of Listeria monocytogenes ATCC® 19111™ and Staphylococcus aureus 3048. Homology-based three-dimensional (3D) structures of SF9C plantaricins PlnJK and PlnEF were predicted using SWISS-MODEL workspace and the helical wheel representations of the plantaricin peptide helices were generated by HELIQUEST. Contrary to the plantaricin-producing SF9C strain, the S-layer-carrying SF9B strain excluded Escherichia coli 3014 and Salmonella enterica serovar Typhimurium FP1 from the adhesion to Caco-2 cells. Finally, PCR-DGGE analysis of the V2-V3 regions of the 16S rRNA gene confirmed the transit of the two selected lactobacilli through the gastrointestinal tract (GIT). Microbiome profiling via the Illumina MiSeq platform revealed the prevalence of Lactobacillus spp. in the gut microbiota of the Lactobacillus-treated rats, even on the 10th day after the Lactobacillus application, compared to the microbiota of the healthy and AlCl3-exposed rats before Lactobacillus treatment. CONCLUSION: The combined application of Lb. plantarum SF9C and Lb. brevis SF9B was able to influence the intestinal microbiota composition in rats, which was reflected in the increased abundance of Lactobacillus genus, but also in the altered abundances of other bacterial genera, either in the model of healthy or aberrant gut microbiota of rats. The antibacterial activity and capacity to withstand in GIT conditions contributed to the functional aspects of SF9C and SF9B strains that could be incorporated in the probiotic-containing functional foods with a possibility to positively modulate the gut microbiota composition.

Transcriptomic and proteomic profiling response of methicillin-resistant Staphylococcus aureus (MRSA) to a novel bacteriocin, plantaricin GZ1-27 and its inhibition of biofilm formation.

Methicillin-resistant Staphylococcus aureus (MRSA) has become a worrisome superbug, due to its wide distribution and multidrug resistance. To characterize effects of a newly identified plantaricin GZ1-27 on MRSA, transcriptomic and proteomic profiling of MRSA strain ATCC43300 was performed in response to sub-MIC (16 µg/mL) plantaricin GZ1-27 stress. In total, 1090 differentially expressed genes (padj < 0.05) and 418 differentially expressed proteins (fold change > 1.2, p < 0.05) were identified. Centralized protein expression clusters were predicted in biological functions (biofilm formation, DNA replication and repair, and heat-shock) and metabolic pathways (purine metabolism, amino acid metabolism, and biosynthesis of secondary metabolites). Moreover, a capacity of inhibition MRSA biofilm formation and killing biofilm cells were verified using crystal violet staining, scanning electron microscopy, and confocal laser-scanning microscopy. These findings yielded comprehensive new data regarding responses induced by plantaricin and could inform evidence-based methods to mitigate MRSA biofilm formation.

Short communication: Antimicrobial potential of Lactobacillus plantarum strains isolated from Slovak raw sheep milk cheeses.

The objective of this study was to screen Lactobacillus plantarum strains isolated from the traditional Slovak raw sheep milk cheese for their inhibitory potential. Seventy-two strains were obtained from samples of raw sheep milk and raw sheep milk cheeses collected from April 2017 to September 2018, in 23 geographical areas of Eastern Slovakia, by inoculation of de Man, Rogosa, and Sharpe agar plates (Oxoid, Basingstoke, UK). Using both the genus- and species-specific PCR methods, 43 strains were identified as Lactobacillus spp., and 10 strains were confirmed as Lb. plantarum. First, the whole bacterial cultures of Lb. plantarum strains were tested by disc diffusion assay. All showed very good antibacterial activities against 6 selected foodborne pathogens, including Escherichia coli, Salmonella Enteritidis, Pseudomonas aeruginosa, Listeria monocytogenes, Staphylococcus aureus, and Bacillus cereus. Then, cell-free neutralized supernatants and partially purified bacteriocins were prepared from the 4 Lb. plantarum strains that exhibited the best antibacterial potential, and they were tested the same way as the whole bacterial cultures. Seven of the 10 Lb. plantarum strains harbored the plnEF gene, 3 strains harbored the plnD gene, and 2 strains possessed both the plnA and plnC genes that encode the production of the respective plantaricins. The presence of both plnR and plnL genes was only detected in a single Lb. plantarum isolate. Based on the results of this study, 4 strains of Lb. plantarum appeared to be suitable candidates for further testing in the dairy manufacturing sector, particularly in the production of raw sheep milk products.

Characterization and evaluation of lactic acid bacteria from indigenous raw milk for potential probiotic properties.

Raw milk contains wide microbial diversity, composed mainly of lactic acid bacteria (LAB), which are used as probiotics in both human and animal husbandry. We isolated, characterized, and evaluated LAB from indigenous Bangladeshi raw milk to assess probiotic potential, including antagonistic activity (against Escherichia coli O157: H7, Enterococcus faecalis, Salmonella Typhimurium, Salmonella Enteritidis, and Listeria monocytogenes), survivability in simulated gastric juice, tolerance to phenol and bile salts, adhesion to ileum epithelial cells, auto- and co-aggregation, hydrophobicity, α-glucosidase inhibitory activity, and antibiotic susceptibility tests. The 4 most promising LAB strains showed probiotic potential and were identified as Lactobacillus casei, Lactobacillus plantarum (which produced plantaricin EF), Lactobacillus fermentum, and Lactobacillus paracasei. These strains inhibited all pathogens tested at various degrees, and competitively excluded pathogens with viable counts of 3.0 to 6.0 log cfu/mL. Bacteriocin, organic acids, and low-molecular-weight substances were mainly responsible for antimicrobial activity by the LAB strains. All 4 LAB strains were resistant to oxacillin and 3 were resistant to vancomycin and streptomycin, with multiple antibiotic resistance indices >0.2. After further in vivo evaluation, these LAB strains could be considered probiotic candidates with application in the food industry.

Antimicrobial activity and preliminary mode of action of PlnEF expressed in Escherichia coli against Staphylococci.

Strains belonging to the genus of Staphylococci, such as Staphylococcus aureus are common pathologic bacteria which may cause nosocomial cross infection and food contamination. Plantaricin EF (PlnEF), a two-peptide nonlantibiotic bacteriocin produced by Lactobacillus plantarum strains, shows great inhibitory effects against Gram-positive Staphylococci strains. To overproduce this two-peptide bacteriocin, plnE and plnF genes were cloned into pET32a (+) vector and heterologously expressed in Escherichia coli by fusion with His6-tag in this study. The purified fusion proteins were cleaved by enterokinase, then PlnE and PlnF peptides without extra amino acids were obtained by a two-step purification method, ultrafiltration centrifuge (10 kDa) followed by a reverse-phase HPLC. Purity of PlnE and PlnF, determined by analytical HPLC, is ∼98%, and their molecular mass confirmed by ESI-MS was 3545.14 Da and 3703.1 Da, respectively. It was found that the two peptides had significant antimicrobial activities against Staphylococcus citreus and Staphylococcus aureus strains and they functioned synergistically. PlnEF exerted its bactericidal activity against Staphylococci strains by permeabilizing the cell membrane, causing influx and efflux of various molecules across the transmembrane barrier, eventually leading to cell death.

Construction, heterologous expression, partial purification, and in vitro cytotoxicity of the recombinant plantaricin E produced by Lactococcus lactis against Enteropathogenic Escherichia coli K.1.1 and human cervical carcinoma (HeLa) cells.

Lactobacillus plantarum produces bacteriocin called plantaricin that can kill or inhibit other bacteria. Plantaricin E (Pln E), a recombinant bacteriocin, has been successfully constructed and produced by a GRAS host, Lactococcus lactis. A polymerase chain reaction (PCR) overlapping technique has been used to construct a ligation of signal peptide gene, Pln A and bacteriocin encoding gene, Pln E. Furthermore, the fusion fragment were cloned into pNZ8148 vector and transformed into L. lactis NZ3900. Molecular expression study shows that recombinant L. lactis NZ3900 is able to express the mature pln E at transcription level with size of 168 bp. Plantaricin E is purified by ammonium sulphate precipitation followed by gel filtration chromatography. Purified fractions were proven to be active against Enteropathogenic Escherichia coli K.1.1. The other fractions of Pln E also have antibacterial activity against several Gram positive and Gram negative bacteria. Purified recombinant plantaricin E is 3.7 kDa in size. The cytotoxicity assay shows purified Pln E inhibits 46.949 ± 3.338% of HeLa cell lines on 10 ppm dose whilst the metabolite inhibits 53.487 ± 2.957% of HeLa cell line on 100 ppm dose. The IC50 calculation of Pln E metabolite is 107.453 ppm, while the purified protein is 11.613 ppm.

Development and antimicrobial application of plantaricin BM-1 incorporating a PVDC film on fresh pork meat during cold storage.

AIMS: The aim of this study was to develop a plantaricin BM-1, a typical IIa bacteriocin produced by Lactocacillus plantarumBM-1, for active polyvinylidene chloride (PVDC) films and to determine the antimicrobial effect of plantaricin BM-1 incorporated into a PVDC film on fresh pork during 7 days of storage at 4°C. METHODS AND RESULTS: Plantaricin BM-1 solutions (20 480 AU ml-1 ) that absorbed into the PVDC film increased gradually and reached maximum volumes during exposure for up to 20 h. When soaked in water, the released amount of plantaricin BM-1 from the active PVDC film reached a maximum at 20 h. The plantaricin BM-1 active PVDC film had an obvious antilisterial effect in culture medium and fresh pork inoculated with Listeria monocytogenes. Furthermore, plantaricin BM-1-incorporated PVDC film was also significantly (P < 0·01) reduced to aerobic counts of approximately 1·5 log10 CFU per g after 7 days of storage at 4°C in pork meat, and the pH and total volatile basic nitrogen of pork meat were significantly (P < 0·01, P < 0·05) lower than those of the control. CONCLUSION: Plantaricin BM-1 active film has an excellent effect to prolong the shelf life of pork meat during cold storage. SIGNIFICANCE AND IMPACT OF THE STUDY: The results of this study suggest a potential application of bacteriocin active film on meat preservation.

Plantaricin NC8 from Lactobacillus plantarum causes cell membrane disruption to Micrococcus luteus without targeting lipid II.

Plantaricin NC8, a two-peptide non-lantibiotic class IIb bacteriocin composed of PLNC8α and PLNC8ß and derived from Lactobacillus plantarum ZJ316, has been shown to be highly potent against a range of bacteria and fungi. In this study, we assessed the antimicrobial mechanism of plantaricin NC8 against the most sensitive bacterial strain, Micrococcus luteus CGMCC 1.193. The results showed that plantaricin NC8 induced membrane permeabilization and caused cell membrane disruption to M. luteus CGMCC 1.193 cells, as evidenced by electrolyte efflux, loss of proton motive force, and ATP depletion within a few minutes of plantaricin NC8 treatment. Furthermore, scanning and transmission electron microscopy showed that plantaricin NC8 had a drastic impact on the structure and integrity of M. luteus CGMCC 1.193 cells. In addition, we found that either PLNC8α or PLNC8ß alone exhibited membrane permeabilization activity, but that PLNC8ß had higher permeabilization activity, and their individual effects were not as strong as that of the combined compounds as plantaricin NC8. Finally, we showed that lipid II is not the specific target of plantaricin NC8 against M. luteus CGMCC 1.193. Our study reveals the antimicrobial mechanism of plantaricin NC8 against M. luteus CGMCC 1.193.