Separation of lactobacilli bacteriocins from fermented broths using membranes

Current separation, isolation and purification techniques to obtain highly potent purified lactobacilli and lactococci bacteriocins include chemical precipitation, separation employing solvents and chromatographic techniques. These methods are arduous, costly, with limited scalability, offering low bacteriocin yields (<20%). To address these challenges, the alternatives of ultrafiltration and nanofiltration, as separation methods were tested. Three promising bacteriocin producing strains, Lactobacillus casei NCIMB 11970, Lactobacillus plantarum NCIMB 8014 and Lactococcus lactis NCIMB 8586 were selected to investigate the applicability and feasibility of the method.To facilitate separation, the microorganisms were grown on specially developed low molecular weight medium (LMWM) mainly containing nutritive sources up to 4 kDa molecular weight. Bacterial cells were removed by centrifugation. The clarified broths were filtered using 4 and 1 kDa MWCO. Bacteriocin activity was determined by an antimicrobial activity test using nisin, which has an inhibitory effect on the growth of susceptible microorganisms. Recovery yields using filtration were found to range between 53 and 68%, a high recovery performance.The bacteriocin activity of crude extracts of all the three lactobacilli were between 95 and 105 IU ml^?1. When the substances were separated using ultrafiltration membrane (4 kDa MWCO) their activity was enhanced to 145–150 IU ml^?1, achieving a total potency yield of 44–53%. Further enhancement of yields up to 36% was attained employing nanofiltration (1 kDa MWCO) membranes with an activity increased up to 200 IU ml^?1.Bacteriocin isolation from crude extracts using filtration was found to be effective, offering high recovery yields, optimising their activity as well as presenting a realistic option towards the formulation of these as commercially available antibacterial agents.     

Purification and Characterization of Bacteriocin Produced by Lactobacillus brevis UN Isolated from Dhulliachar: a Traditional Food Product of North East India

A bacteriocin producing strain Lactobacillus brevis UN isolated from Dulliachar—a salted pickle and identified by biochemical and molecular methods. L. brevis UN was found to produce bacteriocin with broad spectrum activity against spoilage causing/food borne pathogens viz. L. monocytogenes, C. perfringens, S. aureus, L. mesenteroides, L. plantarum and B. cereus. Bacteriocin production was optimized through classical one variable at a time method. The isolate showed maximum bacteriocin production at early stationary phase, pH 4.0, temperature 35 °C and with an inoculum size of 1.5 OD @ 10 %. Bacteriocin produced by L. brevis UN was purified to homogeneity by single step gel exclusion chromatography and was most active at pH 6.0 and 7.0, stable up to 100 °C and was proteinaceous in nature. The results of NMR revealed the presence of proline, glutamic acid, aspartic acid, leucine, isoleucine and serine in its peptide structure. PCR amplification analysis determined that bacteriocin encoded gene in L. brevis UN was plasmid bound.     

Phenotypic and genotypic characterization of bacteriocins in clinical <Emphasis Type="Italic">enterococcal</Emphasis> isolates of Tunisia

The presence of bacteriocin structural genes (entA, entB, entP, entQ, entAS-48, entL50A/B, bac31, and cylL) encoding different bacteriocins (enterocin A, enterocin B, enterocin P, enterocin Q, enterocin AS-48, enterocin L50A/B, bacteriocin 31 and cytolysin L, respectively), and the production of bacteriocin activity were analysed in 139 E. faecalis and 41 E. faecium clinical isolates of Tunisia. Forty-eight of 139 E. faecalis isolates (34%) and 7 of 41 of E. faecium isolates (17%) were bacteriocin producers. Sixty-two per cent of the bacteriocin-producing enterococci showed inhibitory activity against L. monocytogenes. Different combinations of entA, entB, entP, and entL50A/B genes were detected among the seven bacteriocin-producer E. faecium isolates, and more that one gene were identified in all the isolates. The entA gene was associated in most of the cases with entB gene in E. faecium isolates. Cyl _LS were the unique genes detected among E. faecalis (in 24 of 48 bacteriocin-producer isolates, 50%). A β-hemolytic activity was demonstrated in 19 of the 24 cyl _LS -positive E. faecalis isolates (79%), this activity being negative in the remaining five isolates. The presence of different bacteriocin structural genes and the production of antimicrobial activities seems to be a common trait of clinical enterococci.     

Enhanced synthesis and antimicrobial activities of bacteriocins produced by Mexican strains of Bacillus thuringiensis

Recently, we reported the synthesis of five bacteriocin-like inhibitor substances (Bt-BLIS: morricin 269, kurstacin 287, kenyacin 404, entomocin 420, and tolworthcin 524) by Mexican strains of Bacillus thuringiensis. Here we show that, collectively, these Bt-BLIS have a moderate to broad spectrum of antibacterial activity, being toxic to clinically significant against Gram-positive and Gram-negative bacteria, including common etiological agents of human diseases, such as strep throat and scarlet fever, septicemia, pneumonia, urinary tract infection, and emetic and gastrointestinal syndromes. Although synthesis of the five Bt-BLIS was independent of the presence of a target inducing bacterium, we demonstrated for the first time that a proteinaceous component(s) secreted by, or liberated by proteolytic cleavage of Bacillus cereus 183 following treatment with proteinase K, enhanced Bt-BLIS synthesis.     

Intra-specific variation of Lactobacillus plantarum and Lactobacillus pentosus in sensitivity towards various bacteriocins

Fifty-two strains belonging to the Lactobacillus plantarum species group were identified and typed. They represented 32 clones of Lactobacillus plantarum and 7 clones of Lactobacillus pentosus. Sensitivity of all strains towards bacteriocins of four different producer strains was investigated using a deferred inhibition test (DIT). Substantial intra-specific variation in sensitivity of clones was observed towards bacteriocinogenic lactic acid bacteria producing nisin ( Lactococcus lactis ATCC 11454) or pediocin PA-1 ( Pediococcus acidilactici PAC-1.0), while none of the strains were sensitive towards the two remaining bacteriocin producers. The minimum inhibitory concentration (MIC) of nisin towards selected strains confirmed the DIT results. No correlation between the susceptibility of fourteen selected strains towards nisin and an array of antibiotics was found. The present study indicates that the variation in bacteriocin-sensitivity within target species might be a potential limitation for the application of bacteriocins as biopreservatives.     

Sequencing and expression analysis of sakacin genes in Lactobacillus curvatus strains

In this study, we focused our investigation on two strains of Lactobacillus curvatus, L442 and LTH1174, which are able to produce bacteriocins. L. curvatus LTH1174 is widely studied for its capability to produce curvacin A, while L. curvatus L442 was isolated from traditional Greek fermented sausages and was shown to possess a strong inhibitory activity toward Listeria monocytogenes. By polymerase chain reaction, we were able to target in both strains the genes for the production of sakacin P and sakacin Q, sppA and sppQ, respectively, both encoded chromosomally. While sppA was found to be conserved when compared with other sakacin P genes, sppQ showed a deletion of about 15 nucleotides when aligned with sequences obtained from Lactobacillus sakei. This difference did not affect the activity of sakacin Q as determined by testing sensitive strains. Expression analysis highlighted that sakacin P was expressed in L. curvatus L442 but not in L. curvatus LTH1174. Curing experiments were performed on L. curvatus LTH1174 to study the effect of the megaplasmid, present in this strain. In the plasmid-cured strain, expression of the sppA gene was detected. sppQ was expressed in both plasmid-cured and wild-type L. curvatus LTH1174, although expression was higher in the plasmid-cured strain.     

Families of Proteins Forming Transmembrane Channels

Channel-forming proteins/peptides fall into over 100 currently recognized families, most of which are restricted to prokaryotes or eukaryotes, but a few of which are ubiquitous. These proteins fall into three major currently recognized classes: (i) α-helix-type channels present in bacterial, archaeal and eukaryotic cytoplasmic and organellar membranes, (ii) β-barrel-type porins present in the outer membranes of Gram-negative bacterial cells, mitochondria and chloroplasts, and (iii) protein/peptide toxins targeted to the cytoplasmic membranes of cells other than those that synthesize the toxins. High-resolution 3-dimensional structural data are available for representative proteins/peptides of all three of these channel-forming types. Each type exhibits distinctive features that distinguish them from the other channel protein types and from carriers. Structural, functional, and evolutionary aspects of transmembrane channel-formers are discussed. Received: 10 September 1999/Revised: 11 February 2000     

Identification & Characterization of lactobacillus salavarius bacteriocins and its relevance in cancer therapeutics

Therapeutic agents with a goal to eradicate cancer needs to capable of inhibiting the growth and kill, any preformed tumor and should also inhibit oncogenic transformation of normal cells to cancer cells. Bacteriocins are bacterial proteins produced to prevent the growth of competing microorganisms in a particular biological niche and have been proved to possess antineoplastic activity. The entire genome of Lactobacillus salavarius was scanned for putative bacteriocins and subsequently these bacteriocins were characterized by subjecting them as functional annotation algorithms. Azurin is a well characterized bacteriocins with proven cytostatic and apoptotic effect against human cancer cell and was taken as control. Functional characterization revealed that the three bacteriocins Lsl_003, Lsl_0510, Lsl_0554 possessed functional properties very similar to that of Azurin. Molecular screening of these bacteriocins against the common cancer targets p53, Rb1 and AR revealed that Lsl_0510 possessed highest binding affinity towards the all the three receptors making it to ideal candidate for future cancer therapeutics. ABBREVIATIONS: P53 - Protein 53, Rb1 - Retinoblastoma 1, AR - Androgen Receptor, Lsl - Lactobacillus salavarius.     

Cold-adapted microorganisms as a source of new antimicrobials

Thirty out of 8,000 different colony morphotypes isolated from soil samples of Isla de los Estados were selected based on their ability to produce antimicrobials. The significant influence of culture media and incubation temperature on antimicrobial production was proved, being LB medium and 8°C the conditions of choice. Most of the psychrotolerant isolates were phylogenetically related to Serratia proteamaculans (96.4–97.9%) while the psychrophilic isolated 8H1 was closely related to Pseudomonas sp. (90–94% similarity). Produced antimicrobials showed a promising wide spectrum of activity both against gram-positive and gram-negative pathogenic bacteria. They were suspected to be microcin-like compounds (Mw <2,000 Da) and showed a marked tolerance to heat (1 h in boiling water bath) and pH-treatments (1–12). Antimicrobial compounds also showed to partially keep their activity even after overnight freezing at −20 and −80°C and displayed a negative net charge at pH 8.0, a common feature of class II microcins.     

Protein expression vector and secretion signal peptide optimization to drive the production, secretion, and functional expression of the bacteriocin enterocin A in lactic acid bacteria

Replacement of the leader sequence (LS) of the bacteriocin enterocin A (LS_entA) by the signal peptides (SP) of the protein Usp45 (SP_usp45), and the bacteriocins enterocin P (SP_entP), and hiracin JM79 (SP_hirJM79) permits the production, secretion, and functional expression of EntA by different lactic acid bacteria (LAB). Chimeric genes encoding the SP_usp45, the SP_entP, and the SP_hirJM79 fused to mature EntA plus the EntA immunity genes (entA + entiA) were cloned into the expression vectors pNZ8048 and pMSP3545, under control of the inducible P_nisA promoter, and in pMG36c, under control of the constitutive P₃₂ promoter. The amount, antimicrobial activity, and specific antimicrobial activity of the EntA produced by the recombinant Lactococcus lactis, Enterococcus faecium, E. faecalis, Lactobacillus sakei and Pediococcus acidilactici hosts varied depending on the signal peptide, the expression vector, and the host strain. However, the antimicrobial activity and the specific antimicrobial activity of the EntA produced by most of the LAB transformants was lower than expected from their production. The supernatants of the recombinant L. lactis NZ9000 (pNZUAI) and L. lactis NZ9000 (pNZHAI), overproducers of EntA, showed a 1.2- to 5.1-fold higher antimicrobial activity than that of the natural producer E. faecium T136 against different Listeria spp.