Academic literature on the topic 'Plantaricine'

ABSTRACT Plantaricin EF and JK are both two-peptide bacteriocins produced byLactobacillus plantarum C11. The mechanism of plantaricin EF and JK action was studied on L. plantarum 965 cells. Both plantaricins form pores in the membranes of target cells and dissipate the transmembrane electrical potential (Δψ) and pH gradient (ΔpH). The plantaricin EF pores efficiently conduct small monovalent cations, but conductivity for anions is low or absent. Plantaricin JK pores show high conductivity for specific anions but low conductivity for cations. These data indicate that L. plantarum C11 produces bacteriocins with complementary ion selectivity, thereby ensuring efficient killing of target bacteria.

Lactobacillus plantarum 8P-A3 is a strain which is well known on the Russian pharmaceutical market and it is included in several probiotic products. The strain has been widely used since 1973 but the mechanisms of its antibacterial activity were unknown. L. plantarum 8P-A3 expressed high antagonistic activity against the wide range of bacterial pathogens including Streptococcus agalactiae, Streptococcus pyogenes, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Klebsiella pneumoniae strains. DNA sequence analysis of L. plantarum 8P-A3 genome revealed the presence of a complete plantaricin locus of about 20,000 bp encoding genes of at least two bacteriocins – plantaricins EF and NC8. The plantaricin locus found in L. plantarum 8P-A3 is homologous to the plantaricin cluster in L. plantarum J51.

Lactobacillus plantarum is widely found in either anaerobic plant matter or fermented foods, and it has been recognized as producing antimicrobial bacteriocins. This study aimed to characterize the antimicrobial bacteriocins of L. plantarum and detect its genes that encode plantaricins. Samples were isolated from traditional fermented foods from Indonesia. Antimicrobial activity was evaluated using the agar diffusion assay procedure. The titration method applied the maximum amounts of lactic acid at 1054 mg/mL and hydrogen peroxide at 3.85 mg/mL. Based on the results, the supernatant of the L. plantarum strains appeared to have a broad spectrum of antimicrobial activity against pathogens, which would be active at pH 2.0–12.0 and stable temperature. In addition, almost all of the L. plantarum strains contained plantaricin-encoding genes (e.g. plnA, plnF,plnJK, and plnW), which were grouped into one cluster as indicated by phylogenetic analysis. Therefore, this study discovered clear evidence of the potential of some L. plantarum strains to act as antimicrobial agents.

Naturally preservative has potential to appllied as food preservative. A biopreservative can used for food is bacteriocin. Bacteriocin is peptide compound produced by lactic acid bacteria and has antimicrobial activity. Lactobacillus plantarum is one of lactic acid bacteria produce bacteriocin called plantaricin. Plantaricin has antimicrobial activity against pathogen bacteria. Aplication of plantaricin will become an alternative for developing biotechnology in biopreservative. The aim of this research was to evaluate the effectiveness of plantaricin IIA-1A5 as biopreservative to physical properties and microbiological chicken meat at refrigerator temperature. There were two methods used, purification of plantaricin and its aplication to chicken meat. This research used completely randomized design with factorial pattern, then Duncan Multiple Rank Test. The results showed that there was a significant (P<0.05) interaction of gived plantarisin with specific storage time (0, 2, 4, 6, 8, and 10 days) in chicken meat on the quality of meat. As conclusion, plantaricin IIA-1A5 effectively inhibited pathogenic bacteria so total microbe was lower than control. Plantaricin could be recommended as a biopreservative for chicken meat.

Plantaricin IIA-1A5 is a bacteriocin produced by Lactobacillus plantarum IIA-1A5 isolated from Indonesian beef. This research aimed to identify the genes involved in plantaricin IIA-1A5 production and examine its mode of action against Staphylococcus aureus. It has been reported that a bacteriocin structural gene, plnW, is present in genome of L. plantarum IIA-1A5. Here, we reported the presence of additional genes responsible for plantaricin precursor (plnA and plnEF) and a gene encoding the quorum sensor of histidine kinase (plnB). It indicates that genes involved in production of plantaricin IIA-1A5 are organized in at least two bacteriocin operons (plnABCD, plnEFI) and a structural plnW gene. Purified plantaricin IIA-1A5 yielded a single band in SDS-PAGE with apparent size of 6.4 kDa. Amino acid composition of purified plantaricin IIA-1A5 was mainly composed of cationic glutamic acid and cysteine that allowed the formation of disulphide bonds, suggesting plantaricin IIA-1A5 belongs to the pediocin-subclass of class II bacteriocins. Plantaricin IIA-1A5 displayed remarkable antibacterial activity against S. aureus, which was initiated by the adsorption of plantaricin IIA-1A5 onto the cell membrane of S. aureus. The adsorption is hypothesised to be facilitated by non-ionic interactions as it is reduced by the presence of organic solvents or detergents. This adsorption promoted leakage of cellular metabolites through the cell membrane of S. aureus, as indicated by the release of genetic and proteinaceous material of S. aureus observed at 260 and 280 nm, respectively. The leakage also promoted the release of divalent (Ca2+, Mg2+) and monovalent (K+) cations. The release of these intracellular components might be due to pores formed in the cell membrane of S. aureus by plantaricin IIA-1A5 as shown by scanning electron microscopy. Altogether, the mode of action of plantaricin IIA-1A5 against S. aureus seems to be bactericidal as indicated by lysis of the cell membrane.

ABSTRACT Plantaricin BM-1, a typical IIa bacteriocin, is produced by Lactobacillus plantarum BM-1, which can be isolated from a traditionally fermented Chinese meat product. This bacteriocin exhibits perfect thermal stability and broad inhibitory activity against certain foodborne pathogens. In this study, we investigated the effect of plantaricin BM-1 combined with physicochemical treatments (sodium nitrite, heat treatment, ultrahigh pressure technology) on the control of Listeria monocytogenes populations in cooked ham. According to our results, the addition of sodium nitrite (0.075 or 0.15 mg/mL) significantly inhibited the growth of L. monocytogenes in broth, plantaricin BM-1 combined with heat treatment reduced the viable counts of L. monocytogenes more than plantaricin BM-1 alone, and plantaricin BM-1 combined with sodium nitrite and heat treatment had the greatest antibacterial effect in broth. However, the addition of sodium nitrite or heat treatment alone did not inhibit the growth of L. monocytogenes in cooked ham. The combined application of 5,120 arbitrary units per gram (AU/g) plantaricin BM-1 and 0.075 mg/g sodium nitrite reduced L. monocytogenes significantly more effectively (by 1.97 log CFU/g) than application of 5,120 AU/g plantaricin alone. Plantaricin BM-1 application combined with sodium nitrite and heat treatment led to viable counts of L. monocytogenes below the level of detection for 49 days of storage. Moreover, in cooked ham treated with 5,120 AU/g plantaricin BM-1, 0.075 mg/g sodium nitrite, and ultrahigh pressure technology (400 MPa for 5 min), viable counts of L. monocytogenes were reduced by 5.79 log CFU/g compared with control samples at the end of storage. In conclusion, the combination of plantaricin BM-1 with physicochemical treatments had a synergistic inhibitory effect on L. monocytogenes in cooked ham.

The aim of the present study was to assess the technological and safety potential of 207 lactic acid bacteria (LAB) and 195 yeast strains isolated from spontaneously fermented Greek wheat sourdoughs. More accurately, the amylolytic, proteolytic, lipolytic, phytase and amino acid decarboxylase activities, along with the production of exopolysaccharides and antimicrobial compounds by the LAB and yeast isolates, were assessed. A well diffusion assay revealed seven proteolytic LAB and eight yeast strains; hydrolysis of tributyrin was evident only in 11 LAB strains. A further Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) indicated partial hydrolysis of gluten. Lipolysis kinetics over 21 days was applied, exhibiting that lipolytic activity ranged from 6.25 to 65.50 AU/mL. Thirteen LAB inhibited Penicillium olsonii and Aspergillus niger growth and 12 yeast strains inhibited Pe. chrysogenum growth. Twenty-one Lactiplantibacillus plantarum strains exhibited inhibitory activity against Listeria monocytogenes, as well as several sourdough-associated isolates. The structural gene encoding plantaricin 423 was detected in 19 Lcb. plantarum strains, while the structural genes encoding plantaricins NC8, PlnE/F, PlnJ/K, and S were detected in two Lcb. plantarum strains. None of the microbial strains tested exhibited exopolysaccharide (EPS) production, amino acid decarboxylase, amylolytic or phytase activity. The technological and safety potential of the Lcb. plantarum and Wickerhamomyces anomalus strains was highlighted, since some of them exhibited proteolytic, lipolytic, antibacterial and antimould activities.

AbstractIn this paper, we investigated the production of plantaricin 423, produced by Lactobacillus plantarum 423 in both MRS broth and partially deproteinated MRS with Amberlite XAD-1180. Improved production of plantaricin 423 was observed in MRS broth pre-treated with Amberlite XAD-1180. Precipitation of plantaricin with ammonium sulphate, followed by gel filtration chromatography and subsequent analysis on a modified tricine-SDS PAGE, significantly increased the yield and purity of plantaricin 423. This paper describes a novel method to partially deproteinate growth medium in order to facilitate bacteriocin purification.

ABSTRACT Six bacteriocinlike peptides (plantaricin A [PlnA], PlnE, PlnF, PlnJ, PlnK, and PlnN) produced by Lactobacillus plantarumC11 were detected by amino acid sequencing and mass spectrometry. Since purification to homogeneity was problematic, all six peptides were obtained by solid-phase peptide synthesis and were tested for bacteriocin activity. It was found that L. plantarum C11 produces two two-peptide bacteriocins (PlnEF and PlnJK); a strain-specific antagonistic activity was detected at nanomolar concentrations when PlnE and PlnF were combined and when PlnJ and PlnK were combined. Complementary peptides were at least 103times more active when they were combined than when they were present individually, and optimal activity was obtained when the complementary peptides were present in approximately equal amounts. The interaction between complementary peptides was specific, since neither PlnE nor PlnF could complement PlnJ or PlnK, and none of these peptides could complement the peptides constituting the two-peptide bacteriocin lactococcin G. Interestingly, PlnA, which acts as an extracellular signal (pheromone) that triggers bacteriocin production, also possessed a strain-specific antagonistic activity. No bacteriocin activity could be detected for PlnN.

Two application methods were used to investigate the effect of plantaricin BM-1 on the control of Listeria monocytogenes and background spoilage bacteria in sliced vacuum-packaged cooked ham without the addition of any chemical preservatives, including sodium nitrite, during 35 days of storage at 4°C. Regardless of the application method, plantaricin BM-1 treatment (320, 640, or 1,280 arbitrary units [AU]/g of sliced cooked ham) significantly (P &lt; 0.05) reduced the survival of L. monocytogenes (inoculated at 4 log CFU/g of sliced ham) compared with its survival in the control during the first 21 days of storage at 4°C. The inhibitory effect of plantaricin applied to the surface of the ham was significantly better than the same concentration of plantaricin incorporated into the cooked ham (P &lt; 0.0001) during storage. Even 320 AU/g plantaricin applied to the surface exhibited greater inhibition of L. monocytogenes than 1,280 AU/g plantaricin incorporated into the cooked ham on days 1, 14, and 28. A level of 1,280 AU/g plantaricin applied to the surface of the ham reduced L. monocytogenes counts to below the detection limit from the 1st to the 21st day of storage at 4°C. Afterwards, L. monocytogenes was able to regrow, and the viable counts of L. monocytogenes at the end of storage reached 2.76 log CFU/g (6.11 log CFU/g lower than in the control). In the control ham, the counts of background spoilage bacteria increased gradually and surpassed the microbiological spoilage limitation level on the 21st day of storage. However, plantaricin BM-1 treatment significantly (P &lt; 0.05) reduced the survival of background spoilage bacteria in ham compared with their survival in the control from day 21 to 35 of storage at 4°C. A level of 1,280 AU/g plantaricin incorporated into cooked ham was the most effective, reducing the count of background spoilage bacteria count from an initial 2.0 log CFU/g to 1.5 log CFU/g on day 7. This was then maintained for another 14 days and finally increased to 2.76 log CFU/g at the end of the storage at 4°C (2.85 log CFU/g lower than in the control). In conclusion, plantaricin BM-1 application inhibited the growth of L. monocytogenes and background spoilage bacteria in cooked ham during storage at 4°C and could be used as an antimicrobial additive for meat preservation.

Un criblage de 106 isolats de lactobacillus plantarum provenant de divers produits alimentaires, a permis d'en retenir 5 qui manifestaient un effet inhibiteur sur la croissance d'autres bactéries lactiques. Seules trois de ces substances font l'objet de cette thèse (plantaricines SA6, B31 et LC74). Le spectre d'activité de la plantaricine SA6 est un peu plus large que celui des plantaricines B31 et LC74. La production des trois bactériocines est liée à la croissance des bactéries productrices. Les trois plantaricines sont inactivées par les enzymes protéolytiques; cependant, seule l'activité de la plantaricine SA6 n'est pas détruite par les enzymes lipolytiques et glycolytiques. Alors que les plantaricines SA6, B31 et LC74 sont acidotolérantes, seules les deux premières sont relativement thermostables. Plusieurs mutants de lb. Plantarum SA6 et B31 ayant perdu totalement ou partiellement la production des plantaricines SA6 et B31 ont été obtenus après curage plasmidique. Les plantaricines SA6 et B31 ont été totalement purifiées selon deux schémas différents; la plantaricine LC74 ne l'a été que partiellement. La plantaricine SA6 s'absorbe specifiquement puis provoque une lyse cellulaire avec un rélargie des molécules intracellulaires (molécules absorbant dans l'U. V. ; ions potassium) et une augmentation de la perméabilité membranaire à l'ONPG. Des mutants spontanés de listeria monocytogenes ATCC 15313 résistant à trois autres bactériocines découvertes au laboratoire (mesenterocine 52, plantaricine C19 et curvaticine 13) ont été sélectionnés. Stables pendant plusieurs générations, ces mutants apparaissent à une fréquence de l'ordre de 10⁴et manifestent une résistance commune aux trois bactériocines mais non à la nisine

Thesis (MSc) -- University of Stellenbosch, 2004.
ENGLISH ABSTRACT: Lactic acid bacteria play an essential role in the majority of fermented foods by producing organoleptic compounds and increasing the shelf life. The best-studied antimicrobial compounds are bacteriocins, i.e. ribosomally synthesized peptides. Most of these peptides have a narrow spectrum of activity and are usually only active against bacteria from the same ecological niche. The fact that all bacteriocins are degraded by proteolytic enzymes enlarges their potential use as natural food preservatives. The ideal would be to replace or reduce chemical preservatives such as sulfur dioxide, nitrates and nitrites. Bacteriocins are classified into four groups according to their structural and functional characteristics. Plantaricin 423, produced by Lactobacillus plantarum 423, is heat stable, plasmid encoded, relatively small (3.5 kDa) and is classified as a class Iia bacteriocin. The peptide is active from pH 1.0 to 10.0 and inhibits Gram-positive bacteria, including Lactobacillus spp., Leuconostoc spp., Oenococcus oeni, Pediococcus spp., Enterococcus spp., Propionibacterium spp. and pathogens such as Bacillus cereus, Clostridium spp. and Listeria monocytogenes. Production of bacteriocins may occur constitutively or may be regulated by a cell-density dependent system called quorum sensing. Plantaricin 423 is produced throughout logarithmic growth, with no apparent change in production levels when the producer strain is cultured in the presence of plantaricin 423 or Listeria innocua and Lactobacillus sakei. This led us to believe that plantaricin 423 may be produced constitutively. A reporter system was constructed which consisted of the plantaricin 423 promoter, P423, fused to the luxAB genes and cloned into a shuttle vector, pTRKH2. The newly constructed plasmid, pTAB4, was transformed to a bacteriocin-negative mutant of L. plantarum (423 B} Despite several repeats, no luciferase activity was recorded and no RNA homologous to the luxAB genes was detected. The region necessary for expression of plantaricin 423 may be located stream-up of the -80 region homologous to the -80 and -40 conserved repeats of regulated class II bacteriocins. Inclusion of the latter region in the reporter construct may result in the successful expression of luxAB.
AFRIKAANSE OPSOMMING: Melksuurbakteriee speel 'n belangrike rol in die meeste gefermenteerde voedselsoorte deur die produksie van organoleptiese komponente en die verlenging van rakleeftyd. Van aile antimikrobiese komponente is bakteriosiene (ribosomaal gesintetiseerde peptiede) die beste bestudeer. Hierdie peptiede het gewoonlik 'n nou spektrum van antimikrobiese werking en is meestal aktief teen bakteriee in dieselfde ekologiese nis. Die feit dat bakteriosiene deur proteolitiese ensieme in die spysverteringskanaal vernietig word, verhoog die potensiele gebruik van bakteriosiene as preserveermiddels. Die ideaal sal wees om die konsentrasie van chemiese preserveermiddels soos swaweldioksied, nitrate en nitriete te verlaag of rnoontlik te vervang met bakteriosiene. Bakteriosiene word in vier groepe op grond van hul strukturele en funksionele karaktereienskappe geklassifiseer. Plantarisien 423, geproduseer deur Lactobacillus plantarum 423, is hitte-stabiel, word deur 'n plasmied gekodeer, is relatief klein (3.5 kDa) en sorteer onder die klas Iia bakteriosiene. Die peptied is aktief oor 'n wye pH-reeks (pH 1.0-10.0) en inhibeer Gram-positiewe bakteriee, insluitend Lactobacillus spp., Leuconostoc spp., Oenococcus oeni, Pediococcus spp., Enterococcus spp., Propionibacterium spp. en patogene soos Bacillus cereus, Clostridium spp. en Listeria monocytogenes. Produksie van bakteriosiene kan konstitutief plaasvind of kan gereguleer word deur 'n seldigtheids- afhanklike sisteem naamlik "quorum sensing". Plantarisien 423 word regdeur logaritmiese groei geproduseer, met geen verandering in produksievlakke wanneer die produserende stam in die teenwoordigheid van plantarisien 423 of Listeria innocua en Lactobacillus sakei gekweek word nie. Dit het gelei tot die hipotese dat plantarisien 423 moontlik konstitutief geproduseer word. 'n Verklikkersisteem bestaande uit 'n fusie van die plantarisien 423 promoter, P423, aan die luxAB gene is gekonstrueer en in die pendelplasmied pTRKH2 gekloneer. Die nuutgekonstrueerde plasmied, pTAB4, is na 'n bakteriosien-negatiewe mutant van L. plantarum (stam 423 B-) getransfonneer. Ten spyte van etlike herhalings kon geen lusiferase-aktiwiteit opgespoor word nie en kon ook geen homologie in die RNA met die luxAB gene opgespoor word nie. Dit is moontlik dat die area nodig vir uitdrukking van plantarisien 423 verder stroom-op van die -80 area, homoloog aan die -80 en -40 gekonserveerde herhalings van reguleerbare klas II bakteriosiene, gesetel is. Insluiting van laasgenoemde area in die verklikker-konstruk mag lei tot die suksesvolle uitdrukking van luxAB.

Peptídeos antimicrobianos são vistos como alternativas promissoras a serem empregadas pela iindústria farmacêutica no controle de infecções causadas por microrganismos, como também na indústria alimentícia, onde podem desempenhar papéis como conservantes naturais de alimentos. Plantaricina149 é um membro deste grupo, sendo composto por 22 resíduos de aminoácidos, com natureza catiônica e atividade inibitória sobre algumas bactérias patogênicas. Neste trabalho, foram sintetizados diferentes peptídeos análogos à Plantaricina149 para investigar suas ações sobre microrganismos (bactérias e fungos), a fim de correlacionar estes estudos com a ação lítica do peptídeo em modelos de membrana diversos (monocamadas e vesículas fosfolipídicas). A interação de Plantaricina149 com estes sistemas foi monitorada pelas espectroscopias de dicroísmo circular e fluorescência, ensaios de tensão superficial, calorimetria e ressonância plasmônica de superfície, e mostrou ser altamente específica para superfícies fosfolipídicas que apresentam densidade de cargas negativas, tais como a membrana celular de bactérias. A interação eletrostática inicial que se estabelece entre o peptídeo e os fosfolipídios é de extrema importância, sendo capaz de induzir uma estruturação helicoidal na região C-terminal do peptídeo, enquanto a região Nterminal contribui com as interações hidrofóbicas necessárias para a penetração do peptídeo nas camadas fosfolipídicas levando a ruptura das mesmas. De forma semelhante, a atividade antimicrobiana de Plantaricina149a (e alguns de seus análogos) também mostrou ser resultado das interações das duas regiões da molécula, e foi afetada com a retirada ou modificação da região N-terminal do peptídeo. Com a deleção desta região, o peptídeo passou a ter somente ação bacteriostática sobre Staphylococcus aureus e Pseudomonas aeruginosa, perdendo a capacidade bactericida.
Antimicrobial peptides are seen as promising alternatives to be employed in pharmaceutical industry for controlling infections caused by microorganisms, and also in food industry, where they can play roles as natural food preservatives. Plantaricina149 is a member of this group, constituted of 22 amino acid residues, cationic in nature and presenting inhibitory activity against some pathogenic bacteria. In this work, different Plantaricina149 analog peptides were synthesized to investigate their action against microorganisms (bacteria and fungi), with the aim of correlating these studies with the lytic action of the peptide on several membrane models (phospholipid monolayers and vesicles). The Plantaricina149 interaction with these systems was monitored by circular dichroism and fluorescence spectroscopies, surface tension assays, calorimetry and surface plasmon resonance, and showed to be highly specific to phospholipid surfaces that present negative charge density, such as the bacteria cell membrane. The initial peptide-phospholipids electrostatic interaction is extremely important, and it is capable of inducing a helical structure in the peptide C-terminal region, while the Nterminal region contributes with the hydrophobic interactions needed to the peptide penetration in the phospholipid layers and to the disruption of them. Similarly, the Plantaricina149 antimicrobial activity has also proved to be a result of the interactions from the two regions of the molecule, and it was strongly affected by the removal or modification of the peptide N-terminal region. Promoting the deletion of this region has left the peptide only with a bacteriostatic action against Staphylococcus aureus and Pseudomonas aeruginosa, removing its bactericide ability.

106 isolats de Lactobacillus plantarum, provenant de différents produits alimentaires, ont été testés pour leur capacité à produire des bactériocines. La plantaricine C19 de Lactobacillus plantarum C19 isolé de concombres fermentés, possède un large spectre d'action (inhibition de certaines bactéries gram-positives pathogènes et d'altération). La plantaricine V36, produite par Lactobacillus plantarum V36 isolé de vin de palme camerounais, manifeste une action inhibitrice seulement contre des bactéries lactiques. La production de la plantaricine C19 est influencée par la température, le type de milieu de culture, la présence de chlorure de sodium et la nature de l'hydrate de carbone ajouté dans le milieu MRS. L’adsorption des deux plantaricines sur les cellules bactériennes à gram positif est non spécifique. Dans le cas de la plantaricine C19 cette adsorption est fonction du ph ; elle est réduite en présence de sels et après prétraitement des cellules de Listeria grayi CIP 6818 par la lipase et le lysozyme. L’addition d'acide lipoteichoïque dans le milieu réactionnel l'inhibe complètement. Les plantaricines C16 et V36 exercent un mode d'action apparemment bactériostatique en milieux de culture. Des variants résistants de Listeria grayi CIP 6818, présentent une résistance croisée à trois autres bactériocines anti-listeria, mais non à la nisine. Des variants de Lactobacillus plantarum C19, obtenus par curage à la novobiocine, perdent leur capacité à produire la bactériocine et deviennent sensibles à celle-ci. La plantaricine C19 a été purifiée par deux méthodes. L’analyse par spectrométrie de masse (3,851 kDa) et la composition en acides aminés révèlent la présence de 37 à 38 acides aminès. Le séquençage du cote N-terminal a permis l'identification d'une chaine de 28 acides aminés, montrant l'originalité de cette bactériocine

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CNPq
Peptídeos antimicrobianos são componentes importantes do sistema de defesa de diversos organismos contra possíveis invasores. Em geral, são pequenos (até 100 aminoácidos), catiônicos e anfipáticos. Eles têm despertado o interesse da comunidade científica por sua capacidade de atuação contra micróbios, que não conseguem desenvolver resistência a esses peptídeos. Ou seja, eles emergem como complemento e/ou alternativa ao uso dos antibióticos convencionais. Este trabalho desenvolveu um modelo computacional de um peptídeo híbrido de pediocina A (N-terminal) e plantaricina 149a (C-terminal), dois peptídeos bactericidas. Dados experimentais obtidos pelo grupo da prof. Dra. Rosângela Itri do IFUSP foram utilizados para modelagem e comparação dos resultados. Foram feitas simulações de MD do peptídeo interagindo com membranas puras e mistas de POPC e POPG utilizando os parâmetros do campo de força GROMOS 53A6 e 54A7. As simulações com uma unidade do peptídeo revelaram a atualização 54A7 era a mais adequado para modelagem desses sistemas. Os mapas de estrutura secundária mostraram que o peptídeo adquire configuração mais ordenada quando interage com membranas com maior quantidade de POPG em sua composição. As simulações com duas unidades do peptídeo sugeririam que o peptídeo interage e penetra na camada de POPG através da região Cterminal. Na simulação com membrana de POPC, nenhuma das porções terminais ficou estável no interior da membrana. O efeito do aumento da concentração de peptídeos foi examinado colocando cinco e dez unidades do peptídeo para interagir com as membranas. Na membrana de POPC, os peptídeos não formam um único aglomerado e causam pouca perturbação na bicamada. Já na membrana de POPG, o efeito da interação do aglomerado de peptídeos é acentuado, provocando grandes deformações na bicamada lipídica, praticamente a destruindo. Esse fenômeno sugere um possível mecanismo carpete para ação do peptídeo sobre a membrana fosfolipídica de bactérias.
Antimicrobial peptides are important components of defense system in various organizations against possible invaders. They are generally small (100 aminoacids), cationic and amphipathic. They have stimulated the interest of the scientific community for its ability to act against microbes that cannot develop resistance to these peptides. That is, they emerge as complement and/or alternative to the use of conventional antibiotics. This study developed a computational model of a hybrid peptide pediocin A (Nterminal) and plantaricin 149a (C-terminal), two bactericidal peptides. Experimental data obtained by the group of prof. Dr. Rosângela Itri (IFUSP) were used for modeling and compare the results. MD simulations were made of the peptide interacting with pure and mixed POPC and POPG membranes. These simulations were performed using the parameters of the force field GROMOS 53A6 and 54A7. Simulations with a single copy of the peptide revealed that the force field 54A7 was the most appropriate for modeling these systems. The secondary structure maps showed that the peptide acquires a more ordered configuration when interacting with membranes with higher amounts of POPG in its composition. The simulations with two copies of the peptide suggest that the peptide interacts and penetrates the POPG layer via the C-terminal part. In the simulation with POPC membrane, none of the end portions remained stable within the membrane. The effect of increasing the peptide concentration of was examined by placing five and ten copies of the peptide to interact with the membranes. In the POPC membrane, the peptides do not form a single cluster and they cause little disturbance in the bilayer. In the POPG membrane, the interaction of peptides cluster is enhanced, causing large deformation and practically destroying the lipid bilayer. This phenomenon suggests a possible carpet mechanism of action of the peptide on the phospholipid membrane of bacteria.

Thesis (MSc)--Stellenbosch University, 2001.
ENGLISH ABSTRACT: Lactic acid bacteria are present in many foods and beverages and are used as starter cultures in the production of a variety of fermented products. Many of these bacteria produce ribosomally synthesized antimicrobial peptides (bacteriocins), which inhibit the growth of bacteria genetically closely related to the producer cell. Since many of these target bacteria include foodbome pathogens such as Bacillus spp., Clostridium spp., Listeria spp., and Staphylococcus spp., the practical importance of these peptides as food preservatives has been well documented and, in the case of nisin and pediocin PA-I, commercially explored. The increased demand from health conscious consumers for foods with no chemical preservatives is putting renewed pressure on the producer to supply a "clean and green" product, but with the same or even an extended shelf life. Various research groups are screening lactic acid bacteria for production of novel broad-spectrum antimicrobial peptides or are exploring the possibilities of altering known bacteriocins to inhibit Gram-negative bacteria, yeasts and molds. Pediocin PD-I, produced by Pediococcus damnosus NCFB 1832, belongs to the class Ila bacteriocins, i.e. heat-stable Listeria-active peptides, containing the YGNGV -consensus sequence in the N-terminal region. Little is known about the production and mode of activity of pediocin PD-I. In this study, production of pediocin PD-I was significantly increased by optimizing the growth medium, De Man Rogosa and Sharpe (MRS) broth. Addition of bacteriological peptone (1.7%, w/v), manganous sulphate (0.014%, w/v) and Tween 80 (3%, v/v), and lowering of the pH during fermentation stimulated pediocin PD-I production and the level of organic acids produced. Maximum levels of bacteriocin activity were recorded at an initial pH of 6.7 in the latter medium. Under these conditions the specific bacteriocin activity increased by a factor of approximately six after 55 h of fermentation. The effect of pediocin PD-I, plantaricin 423, produced by Lactobacillus plantarum 423, and commercial grade nisin (Aplin and Barrett Ltd., Trowbrige, Wilts, England) was tested against planktonic cells of Oenococcus oeni and a biofilm of the cells established on stainless steel surfaces identical to those used in wineries. After 5 h of treatment with 3000 AU (arbitrary units )/ml of each bacteriocin, all planktonic cells of 0. oeni in a modified Chardonnay must medium were killed. All viable cells in the biofilm were killed after only 1 h in the presence of 3000 AU/ml of anyone of the bacteriocins. In addition, pediocin PD-I, plantaricin 423 and nisin removed the biofilms from the surfaces and reduced the biomass either completely, as in the case of pediocin PD-I, or by 58% and 50% as in the case of plantaricin 423 and nisin, respectively. These same results were recorded after 5 h of treatment with 3000 AU/ml in a modified Chardonnay must medium. To our knowledge this is the first report of controlling biofilm formation of malolactic bacteria on stainless steel surfaces with natural antimicrobial peptides. This implies that, apart from being very effective in controlling the cell numbers of free-living cells of 0. oeni, the three bacteriocins, especially pediocin PD-I, could also be used as natural sanitizers. The fact that the production and activity levels ofpediocin PD-I could be increased without genetically modifying the producer strain is an added advantage.
AFRIKAANSE OPSOMMING: Melksuurbakterieë is teenwoordig in verskeie soorte voedsel- en drankprodukte en word as suurselkulture in die produksie van 'n verskeidenheid gefermenteerde produkte gebruik. Baie van hierdie bakterieë produseer ribosomaal-vervaardigde antimikrobiese peptiede (bakteriosiene) wat die groei van ander bakterieë, geneties naverwant aan die produserende organisme, inhibeer. Omdat baie van hierdie bakterieë voedselpatogene soos Bacillus spp., Clostridium spp., Listeria spp. en Staphylococcus spp. insluit, is die praktiese belang van hierdie peptiede reeds deeglik ondersoek en word, soos in die geval van nisien en pediosien PA-I, kommersieel gebruik. Die toenemende behoefte van die verbruiker na voedselprodukte met geen chemiese preserveermiddels plaas nuwe druk op die vervaardiger om veilige voedselprodukte te produseer, maar met dieselfde of selfs langer rakleeftyd. Verskeie navorsingsgroepe bestudeer melksuurbakterieë vir die produksie van unieke antimikrobiese peptiede met 'n wye spektrum van inhibisie en ondersoek ook die moontlikhede om hierdie bakteriosiene geneties te manipuleer ten einde Gram-negatiewe bakterieë, giste en swamme te inhibeer. Pediosien PD-l, geproduseer deur Pediococcus damnosus NCFB 1832, word as 'n klass na bakteriosien geklassifiseer. Hierdie groep sluit in die hitte-stabiele Listeria-aktiewe peptiede, met 'n YGNGV-konsensus volgorde in die N-terminale deel van die peptied. Min is egter bekend oor die meganisme van werking van hierdie bakteriosiene. In hierdie studie is die produksie van pediosien PD-l betekenisvol verhoog met die optimalisering van die vloeibare groeimedium De Man Rogosa en Sharpe (MRS). Die toevoeging van bakteriologiese peptone (1.7%, miv), mangaan sulfaat (0.014%, miv) en Tween 80 (3.0%, v/v) en 'n afname in die pH gedurende groei het pediosien PD-l-poduksie gestimuleer en sodoende ook die vlak van organiese sure wat geproduseer is. Maksimum vlakke van bakteriosien-aktiwiteit is in hierdie medium met 'n aanvangs-pH van 6.7 waargeneem. Onder hierdie omstandighede, en na 55 uur van fermentasie, het die spesifieke aktiwiteit van die bakteriosien met 'n faktor van ongeveer ses verhoog. Die effek van pediosien PD-l, plantarisien 423, geproduseer deur Lactobacillus plantarum 423, en 'n kommersiële graad nisien (Aplin and Barrett Ltd., Trowbride, Wilts, Engeland) is teen die planktoniese selle van Oenococcus oeni en 'n biofilm van hierdie selle, gevestig op 'n vlekvrye staaloppervlak identies aan wat in wynkelders gebruik word, getoets. Na 5 ure van behandeling met 3000 AB (arbitrêre eenhede)/ml van elke bakteriosien, is al die planktoniese selle van O. oeni in 'n gemodifiseerde Chardonnay mos-medium vernietig. Alle lewensvatbare selle in die biofilm is ook na slegs 1 uur in die teenwoordigheid van 3000 AE/ml van enige een van hierdie bakteriosiene vernietig. Verdermeer het pediosien PD-I, plantarisien 423 en nisien ook die biofilm op die vlekvrye staal-oppervlak verwyder. In die geval van pediosien PD-I is 'n totale afname van die biomassa-oppervlak waargeneem, terwyl plantarisien 423 en nisien 58% en 50% van die totale biomassa verwyder het. Hierdie resultate is na 5 ure van behandeling (3000 AE/ml) in 'n gemodifiseerde Chardonnay mos-medium waargeneem. Sover ons kennis strek is hierdie die eerste verslag rakende die gebruik van natuurlike antimikrobiese peptiede om biofilm-vorming deur appel-melksuurbakterieë op vlekvrye staal oppervlaktes te beheer. Dit impliseer dat bakteriosiene, spesifiek pediosien PD-I, benewens die beheer van planktoniese selle van appel-melksuurbakterieë, ook as natuurlike oppervlak-reinigers gebruik kan word. Die feit dat die produksie en aktiwiteitsvlakke van pediosien PD-I verhoog kon word sonder om die organisme geneties te modifiseer is 'n verdere voordeel.

碩士
嘉南藥理科技大學
生物科技系暨研究所
98
In this study, 11 isolated strains of lactic acid bacteria were tested antibacterial activity by agar diffusion method, using Escherichia coli, Salmonella spp., Staphylococcus aureus and Streptococcus mutans as indicator strains. Antibacterial test results showed that all 11 lactic acid bacteria have higher inhibitin zone against E. coli and Salmonella spp.. Among of them, 6 strains of lactic acid bacteria; B0013, B0039, B0106, B0125, B0126 and B0134 showed inhibitory activity against all the indicator strains. B0013、B0055、B0105、B0106、B0115、B0125、B0126、B0134、B0157、B0158 were identified as Lactobacillus plantarum and B0039 was Lactobacillus paracasei by 16S rDNA identification. At revealed that the 16S rDNA nucleotide sequence of there isolated strains were of 99% similarity with that of NCBI database. The bacteriocin locus from isolates B0055、B0106、B0115 and B0126 showed remarkable similarity to the plantaricin loci described for Lactobacillus plantarum WCFS1. B0126 was cultured in modified DMB containg 2% of dextrin, the number of colonies were 1.55×109 CFU/ml, similar with the culture of MRS. Further more, in storage test, B0126 supernatant was against S. mutans, the results showed the antibacterial activity of storage time were about 3.5 months. In the bacteriocin induction was observed that B0126 can be induced more bacteriocin against E. coli by BCRC12248.