No description available.

No description available.

The metabolic activity of bacterial microorganisms may influence the growth and metabolic activities of other microbes that are present in any specific niche. Lactic acid bacteria (LAB) are antagonistic to some microbial pathogens by the metabolic production of compounds with antimicrobial activity. Consequently, investigators have measured the effects of those antimicrobials to inhibit specific pathogens. However, the mode(s) of action of LAB against foodborne pathogens on products and/or in broth is not completely understood. Therefore, the objectives of this research were to (i) determine the LAB dose required for inhibition of Escherichia coli O157:H7 and Salmonella enterica in vitro and on spinach, and (ii) identify and quantify the major antimicrobial substances synthesized by LAB as a function of postinoculation storage conditions. Assays were performed at 7 degrees C under aerobic conditions. The foodborne pathogens dose responses were assessed in a liquid microbiological medium (in vitro) and on spinach leaf surfaces. Different levels of foodborne pathogens and LAB cultures were used. The addition of LAB cultures did not reduce E. coli O157:H7 or Salmonella enterica populations when performed in vitro. However, when LAB cultures were sprayed on the surfaces of spinach leaves at 8.0 log10 CFU/g, there were significant reductions on E. coli O157:H7 of 1.62 and 0.73 log10 CFU/g (after 3 days) and on Salmonella enterica of 1.85 and 0.71 log10 CFU/g (after 6 days) for treatments inoculated with an initial level of 2.0 and 4.0 log10 CFU/g, respectively. After quantification of the antimicrobial compounds synthesized by LAB cultures, they were correlated against the population growth of targeted pathogens. The highest Llactic acid (3.71 plus/minus 0.14 micromoles/ml, day 12) and hydrogen peroxide (3.72 plus/minus 3.34 microM, day 6) production were obtained from the in vitro sample inoculated with 8.0 log10 CFU/ml of LAB and 0.0 log10 CFU/ml of pathogens. The highest bacteriocin production (0.1 plus/minus 0.01 mg/ml) was obtained from the in vitro sample with 8.0 log10 CFU/ml of LAB and 2.0 log10 CFU/ml of pathogens. In conclusion, the LAB cultures were able to produce detectable amounts of antimicrobials that may be used as intervention and/or sciencebased practice against foodborne pathogens by producers and the industry.

Enterococcus faecalis 710C is a lactic acid bacterium that produces two bacterocins, ent7A and ent7B. Both ent7A and ent7B have strong activity against gram-positive food pathogens including Listeria spp., Clostridium spp., vancomycin-resistant enterococci (VRE) and methicillin-resistant Staphylococcus aureus (MRSA). Mass spectrometry analyses revealed that both ent7A (5201 Da) and ent7B (5207 Da) have formylated N-terminal methionine. The amino acid sequences, structural gene sequences of ent7 from nucleotide position 1-275 and immunity gene were determined. Circular dichroism data suggest that in aqueous solution ent7A and ent7B have 20 to 25% alpha-helical region. Addition of membrane-mimicking reagent (trifluoroethanol) did not significantly enhance the alpha-helical content in ent7A and ent7B. Chiral analysis by gas chromatography- mass spectrometry showed that the amino acid residues elucidated in ent7A and ent7B were all in L-configuration. / Food Science and Technology

Rapid development of antimicrobial resistance (AMR) among bacteria, combined with diminished new antibiotic discovery rates, is an increasing threat to human health. Bacterially derived antimicrobial peptides (AMP) hold excellent potential as potent novel therapeutics. This study embraces traditional natural AMP discovery methods and the newer in silico genome mining tool BAGEL 3 to facilitate identification of novel antimicrobial agents. The traditional screening efforts led to the discovery of two promising antimicrobial producer strains; Bacillus pumilus J1 producing two AMPs, peptides NI03 and NI04, and Klebsiella pneumoniae A7, which produced peptide NI05. In silico mining of the B. pumilus J1 and K. pneumoniae A7 genomes and those from under exploited anaerobic bacteria using BAGEL 3 yielded 18 putative bacteriocin structures that were associated with multiple known and relevant bacteriocin accessory genes and/or carried significant homologies to known bacteriocins. Peptide NI04 proved to be active against Gram positive species only, including meticillin resistant Staphylococcus aureus and vancomycin resistant enterococci and peptide NI03, in addition to these pathogens, showed activity against E. coli. Peptide NI05 was active against Gram-negative pathogens including extended spectrum β-lactamase producing E. coli. All isolated peptides were observed to be proteinaceous in nature and highly heat stable. Peptides were purified or partially purified using solid phase extraction followed by RP-HPLC. The mass of the peptides was determined using ESI or MALDI-TOF mass spectrometry. Tandem MS fragmentation of peptide NI04 generated several sequence tags. Draft genome sequences of the B. pumilus J1 and K. pneumoniae A7 producer strains were obtained using the Illumina MiSeq platform. This allowed identification of the genes encoding peptide NI04, which was confirmed to be novel and was named pumicin NI04. Further characterisation of pumicin NI04 demonstrated it was non-toxic to keratinocytes, Vero cells and non-haemolytic up to at least 18x the minimum inhibitory concentration. The discovery revealed that pumicin NI04 belongs to the WXG-100 peptide superfamily, having homology with the mycobacterial and staphylococcal virulence factor EsxA. This represents the first report of antimicrobial activity in a WXG-100 peptide and has intriguing evolutionary implications. Although it was not possible to fully characterise peptides NI03 and NI05, when BAGEL 3 was used to mine the B. pumilus J1 genome, a promising putative bacteriocin candidate was identified that was homologous to Enterocin AS-45, which also confers anti Gram-negative activity and may be related to the activity observed for NI03, however more evidence is required. Investigations of the K. pneumoniae A7 bacteriocin on the other hand helped establish that the K. pneumoniae microcin E492 pathway was present and highly conserved in strain A7, and is likely to be responsible for the activity observed indicating that NI05 was not a novel peptide.

Antimicrobial-resistant (AMR) bacteria are a major source of concern in modern-day nosocomial settings, leading to possible further drug resistance or spread to those who cannot fight off the infection. Previous work from our laboratory has shown that Klebsiella pneumoniae (KP) secretes an antimicrobial protein that has been shown to inhibit the growth of many species of bacteria that contain AMR properties in the Enterobacteriaceae family, a major contributor to nosocomial AMR. Klebsiella spent media is able to inhibit the growth of Citrobacter freundii (CF), Enterobacter aerogenes (EA), and Enterobacter cloacae (ECL) through an anti-microbial protein (AMP). This AMP has been shown to reduce the density and growth of CF, EA, and ECL in both biofilm and planktonic forms. To determine the genetic elements involved in AMP production, we introduced a transposon (Tn5) into the genome of Klebsiella to provide resistant selection and to create a mutant knockout to find the exact location of the gene. Upon transposon mutagenesis, the resulting genome was electroporated into Rec- E. coli. The E. coli was now able to produce the antimicrobial protein, with the zones of inhibition for CF, EA, and ECL. Upon confirmation that the plasmid mediates the AMP, the plasmid was sent for sequencing to further characterize the gene responsible for coding the AMP. This newly identified AMP may prove to be a valuable treatment for AMR bacteria once characterized.

Viren haben als Ursache lebensmittelassoziierter Infektionen eine große Bedeutung. Sie können vor allem über rohe oder unzureichend erhitzte Lebensmittel übertragen werden. In diesem Zusammenhang werden grüner Salat, Erdbeeren, Himbeeren, Frühlingszwiebeln, Muscheln, halbgetrocknete Tomaten, fäkal verunreinigtes Trinkwasser, Backwaren und Rohwürste als häufige Infektionsquellen genannt. Vor allem kurzgereifte Rohwürste gehören aus mikrobiologischer Sicht zu Risikoprodukten. Um eine gleichbleibende Qualität der Produkte zu gewährleisten, ist die Verwendung von Starterkulturen unerlässlich. Als sogenannte Schutzkulturen sollen sie gleichzeitig die Vermehrung unerwünschter bakterieller Pathogene unterbinden. Bisher ist allerdings nicht bekannt, inwieweit diese zur Virusinaktivierung in kurzgereiften Rohwürsten führen bzw. beitragen. Aus diesem Grund war es das Ziel dieser Arbeit, den Einfluss von rohwurstrelevanten Starter- und Schutzkulturen sowie deren Metabolite (Bacteriocine, Milchsäure) auf die Tenazität und Inaktivierungskinetik von Viren zu prüfen. Die Untersuchungen erfolgten mit dem murinen Norovirus (MNV) S 99 sowie dem humanen Influenzavirus H1N1 (A/WSN/33). Antivirale Effekte wurden zum einen anhand von in-vitro-Studien, zum anderen anhand von experimentell mit Viren kontaminierten kurzgereiften Rohwürsten (Mettwurst/Teewurst) geprüft. Die Bacteriocine Sakacin A und Nisin zeigten in phosphatgepufferter Salzlösung (PBS) keine viruzide Wirkung gegenüber MNV S 99 und H1N1 (pH 6,2; 24 °C; Exposition: 3 Tage). Weiterhin wurden anhand von in-vitro-Untersuchungen 29 verschiedene zellfreie Kulturüberstände [Milchsäurebakterien, Staphylococcus spp. (S.), Kocuria (K.) varians] hinsichtlich ihrer antiviralen Wirkung geprüft. Dabei konnte eine signifikante Titerreduktion von MNV S 99 bei Exposition mit dem Kulturüberstand eines Lactobacillus (Lb.) curvatus-Isolates festgestellt werden (p < 0,05). In mit dieser Kultur fermentiertem Tee- und Mettwurstbrät zeigte sich jedoch kein Effekt. Die Virustenazität von H1N1 und MNV S 99 konnte mit D,L-Milchsäure unter rohwurstrelevanten Bedingungen (pH 5,0 bis 6,2) sowohl in-vitro als auch im frischen Mettwurstbrät beeinflusst werden. In-vitro erzielte Titerreduktionen lagen bei 2,5 (H1N1) bzw. 3,25 log-Stufen (MNV S 99) nach drei Tagen (24 °C) Lagerung. Im Gegensatz dazu war MNV S 99 im Vergleich zu H1N1 im Mettwurstbrät stabiler. H1N1 konnte unterhalb von pH 5,5 bereits direkt nach dem Einmischen der Influenzaviren in das Wurstbrät nicht mehr nachgewiesen werden. MNV S 99 wurde hingegen erst nach einem Tag Lagerung (22 °C) maximal um 0,7 log-Stufen reduziert (pH 5,2). Die verwendeten Starter- und Schutzkulturen (Lb. sakei, Lb. curvatus, Lb. paracasei, Lb. plantarum, S. carnosus, S. xylosus, K. varians) zeigten im Mett- und Teewurstbrät im Vergleich zur Kontrolle (ohne Starterkultur) keinen zusätzlichen viruziden Effekt auf MNV S 99. Zunehmende Virustiterreduktionen konnten mit pH-Wert-Erniedrigung beobachtet werden. Nach der Reifung (1 Tag, 22 °C, pH 4,9) von Mettwurst mit Starterkulturen wurde das Virus um maximal 1,65 log-Stufen reduziert. In mit Einzel- beziehungsweise Mehrstamm-Mischkulturen fermentierter Teewurst (7 Tage, 22 °C, pH 4,9) betrug die Titerreduktion maximal 1,10 log-Stufen. Das Influenzavirus H1N1 konnte im Rohwurstbrät mit Starterkulturen auch nach Verwendung hoher Ausgangstiter bereits zu Beginn der Untersuchungen nicht mehr nachgewiesen werden. Aus den erzielten Daten kann geschlussfolgert werden, dass die Bacteriocine Sakacin A und Nisin nicht als antivirale Zusatzstoffe in Lebensmitteln (z. B. Rohwürste) geeignet sind. Das antivirale Potential von zellfreien Kulturüberständen war Bakterienstamm-spezifisch und nur in-vitro ersichtlich. Daher muss die Nutzung des Lb. curvatus 1-Stammes nicht anderen rohwurstrelevanten Starterkulturen vorgezogen werden. Die Verwendung von Milchsäure als Zusatzstoff im Rohwurstbrät eignet sich nur zum Ausschluss einer viralen Exposition im Zusammenhang mit H1N1. Frische Mettwurst muss allerdings hierzu adäquat gesäuert (pH < 5,5) werden. Neben dem antiviralen Effekt durch gebildete Säure, konnte keine weitere spezies-spezifische antivirale Wirkung verwendeter Starter- und Schutzkulturen auf MNV S 99 festgestellt werden. Die Säureleistung einzelner Kulturen ist demzufolge für eine Virusinaktivierung entscheidend. Das antivirale Potential verwendeter Starter- und Schutzkulturen in Rohwürsten ist im Zusammenhang mit MNV S 99 als gering einzuschätzen. Unter der Annahme, dass murine und humane Noroviren eine ähnliche Tenazität in kurzgereiften Rohwürsten aufweisen, sollten diese Produkte im Zusammenhang mit Noroviren als Risikoprodukte eingestuft werden.

Thesis (MSc (Institute for Wine Biotechnology))--Stellenbosch University, 2010. / ENGLISH ABSTRACT: The process of winemaking involves a number of microorganisms, contributing both positively and negatively to the final product. Lactic acid bacteria (LAB) are present at all stages of vinification and therefore play a major role in the production of wine, especially red wine. LAB are responsible for malolactic fermentation (MLF), which can be desirable or unwanted depending on the style of wine. LAB can also be responsible for spoilage, and production of off flavours resulting in a decrease in the quality of the finished wine. Spoilage occurs if the wrong species are present at the wrong time and can also occur as a result of spontaneous MLF. It is therefore necessary to control the population of indigenous LAB present in the wine. Plantaricins are bacteriocins produced by Lactobacillus plantarum strains and have the potential to inhibit closely related strains that occupy the same ecological niche. This makes them promising for the control of LAB during the winemaking process. Inhibition of the indigenous LAB microflora could help to prevent the formation of undesirable off-flavours, as well as allowing for control over MLF. The use of plantaricin-producing starter cultures could also lead to a reduction in the amount of sulphur dioxide used in wine. The purpose of this study was to investigate the potential of L. plantarum strains isolated from wine to produce plantaricins under winemaking conditions. This potential was evaluated by investigating the expression of plantaricin genes under winemaking conditions. The first objective was to screen nineteen strains of L. plantarum isolated from South African red wines, as well as a commercial strain; for various genes responsible for the production of plantaricins, including structural, transport and regulatory genes. Results showed that the twenty strains contained at least 16 of the 24 genes (previously reported to be associated with the plantaricin locus for various L. plantarum strains) screened for. Only orfZ123 and orf345 genes yielded no positive results in any of the strains. The second objective was to sequence selected plantaricin genes (plnE, plnF, plnN, plnG and plnB) to determine the variation in nucleotide and amino acid sequences of these genes among the different wine L. plantarum isolates. High homology was found between the nucleotide sequences of the strains and none of the amino acid substitutions in the protein sequences occurred in conserved regions. The nucleotide sequence of plnN was identical in all but one of the strains and similarity of the plnB sequence ranged from 96% to 100%. Similarity of the plnG nucleotide sequence ranged from 99% to 100%. The plnE nucleotide sequence was identical in all but two strains and there were only two groups in terms of nucleotide sequence for plnF, with only two changes between the groups. The third objective was the evaluation of plantaricin production using plate assays mimicking certain wine parameters (pH and ethanol concentration). All twenty strains showed inhibitory activity to varying degrees against a panel of nine indicator microorganisms, including Enterococcus faecalis, Listeria monocytogenes and potential wine spoilage organisms, Lactobacillus spp, Pediococcus spp and Leuconostoc mesenteroides. Addition of 10% ethanol and a low pH of 3.5 decreased both the bacteriocin production as well as the spectrum of activity. Seven of the twenty strains, however, showed good bacteriocin activity under all conditions. The fourth objective was to investigate the expression of two plantaricin structural genes (plnEF and plnJK) and the transporter gene (plnG) under winemaking conditions. Two strains (R1122 and 113.1) were chosen, based on the results from the previous objectives, as starter cultures for MLF in synthetic wine media and Riesling wine. Low wine pH (3.2) and high wine pH (3.8) levels were investigated in conjunction with ethanol concentrations of 0%, 12% and 15%. All three of the genes were expressed to varying degrees depending on the fermentation condition. High ethanol and low pH generally decreased expression of the structural plantaricin genes. The influence on expression of the transporter gene was different, with low pH and presence of ethanol resulting in an increase in gene expression. The genes were also expressed in wine, although at a lower level relative to expression in the synthetic wine media. The presence of sensitive bacteria in the wine seemed to increase expression of the structural genes. Furthermore, expression of the mle gene responsible for MLF was investigated under the same winemaking conditions. Expression was shown to be inducible by malic acid, and negatively affected by the presence of ethanol but positively influenced by a lowering in pH from 3.8 to 3.2. This study confirms that plantaricin genes are expressed under winemaking conditions, which in turn indicates that the plantaricins could be produced under winemaking conditions. This confirms the potential use of these plantaricin-producing strains as starter cultures for MLF with the ability to inhibit indigenous LAB, however, presence of the plantaricin protein in wine still needs to be confirmed. It will also need to be established whether the protein is biologically active and not inhibited by wine-related factors. / AFRIKAANSE OPSOMMING: Die proses van wynmaak bevat 'n verskeidenheid mikroorganismes, wat postiewe en negatiewe bydrae kan lewer tot die finale produk. Melksuurbakterieë is teenwoordig by alle stadiums van wynmaak en speel 'n belangrike rol in die produksie van wyn. Melksuurbakterieë is verantwoordelik vir appelmelksuur gisting (AMG), wat gewens of ongewens kan wees, afhangende van die styl van die wyn. Melksuurbakterieë kan ook verantwoordelik wees vir bederf van wyn, asook die produksie van ongewenste geure wat bydrae tot ʼn toename in die kwaliteit van die wyn. Bederf van wyn kan gebeur as die verkeerde spesies voorkom op die verkeerde tyd en kan ook gebeur as ʼn gevolg van spontane AMG. Dit is dus nodig om die populasie van natuurlike melksuurbakterieë in wyn te beheer. Plantarisiene, geproduseer deur Lactobacillus plantarum wyn-isolate, het die potensiaal om naby verwante stamme se groei te inhibeer wat in dieselfde nis voorkom. Hierdie eienskap maak hul belowend vir die beheer van melksuurbakterieë se groei gedurende die wynmaakproses. Inhibering van die natuurlike mikroflora kan help om die vorming van ongewenste geure te verhoed, sowel as om AMG te beheer. Die gebruik van aanvangskulture, wat plantarisiene kan produseer, kan lei tot ’n vermindering in die gebruik van swaweldioksied in die wynindustrie. Die doel van hierdie studie was om die potensiaal van L. plantarum stamme, geïsoleer vanuit wyn, te ondersoek vir hul vermoë om plantaricins te produseer in toestande wat die wynmaakproses naboots. Die potensiaal was ondersoek deur te kyk na die uitdrukking van plantarisien-produserende gene onder wynmaak toestande. Die eerste objektief was om die 19 L. plantarum stamme, geïsoleer vanuit Suid-Afrikaanse rooi wyne, asook n kommersiele stam, te ondersoek vir die teenwoordigheid van verskeie gene wat verantwoordelik is vir die produksie van plantarisiene, sowel as strukturele, transporter en regulerende gene. Al twintig van hierdie stamme het ten minste 16 uit die 24 gene bevat waarvoor ondersoek was. OrfZ123 en orf345 het egter geen positiewe resultate opgelewer in enige van die stamme nie. Die tweede objektief was om die DNA-volgorde te bepaal van spesifieke gene (plnE, plnF, plnN, plnG, sowel as plnB) en sodoende die variasie in nukleotied en aminosuur volgorde van hierdie gene in die verskillende L. plantarum wyn-isolate te bepaal. Hoë vlakke van homologie was gevind en geen van die aminosuur veranderings het in behoue gebiede plaasgevind nie. Die nukleotied volgorde van plnN was identies in al die stamme, behalwe vir een, en die ooreenkomste tussen die plnB volgorde het varieër van 96% tot 100%. Die ooreenkomste tussen die plnG nukleotied volgorde het varieër van 99% to 100%. Die plnE nukleotied volgorde was identies in al die stamme, behalwe vir twee, en daar was net twee groepe in terme van nukleotied volgorde vir plnF, met net twee veranderinge tussen die groepe. Die derde objektief was om die vermoë van die stamme om plantaricins the produseer, deur gebruik te maak van plaat assays, onder verskillende wyntoestande te ondersoek. Die twinting stamme het verskillende vlakke van inhibering teenoor die nege toets-organismes getoon, wat Enterococcus faecalis, Listeria monocytogenes sowel as potensiele wyn bederf organismes, Lactobacillus spp, Pediococcus spp and Leuconostoc mesenteroides insluit. Die byvoeging van 10% etanol en ’n lae pH van 3.5, het beide bakteriosien produksie inhibeer, sowel as die spektrum van aktiwiteit verminder. Sewe van die stamme het egter steeds goeie aktiwiteit getoon onder al die kondisies wat getoets was. Die vierde objektief was om die uitdrukking van twee plantaricin strukturele gene (plnEF en plnJK), sowel as die transporter geen (plnG) onder wynmaak omstandighede te ondersoek. Twee stamme (R1122 en 113.1) was gekies as aanvangskulture vir AMG in sintesiese wyn media, sowel as Riesling wyn. Hierdie twee stamme was gekies op grond van die resultate wat van die vorige objektiewe verkry was. Lae wyn pH (3.2) en hoë wyn pH (3.8) was ondersoek in samewerking met verskillende etanol konsentrasies wat 0%, 12% en 15% etanol insluit. Al drie hierdie gene was uitgedruk teen verskillende vlakke, afhangende van die verskeie fermentasie kondisies. Hoë etanol en lae pH lei oor die algemeen tot ʼn toename in uitdrukking van die strukturele plantarisien gene. Die invloed op uitdrukking van die transporter geen was verskillend, want lae pH en die teenwoordigheid van etanol het gelei tot ʼn verhoging in geen uitdrukking. Die gene was uitegdruk in wyn, maar was teen laer vlakke relatief tot uitdrukking in die sintetiese wyn media. Dit blyk dat die teenwoordigheid van sensitiewe bakterieë in die wyn tot ‘n hoër uitdrukking van die strukturele gene lei. Die uitdrukking van die mle geen, verantwoordelik vir AMG, was ook onder dieselfde wynmaak kondisies ondersoek. Die uitdrukking was geïnduseer deur appelsuur, negatief beïnvloed deur die teenwoordigheid van etanol, maar positief beïnvloed deur ’n verlaging in pH van 3.8 tot 3.2. Hierdie studie toon dat plantaricin gene uitegedruk word onder wynmaak toestande en dat plantaricins moontlik onder hierdie toestande geproduseer kan word. Die potensiaal van hierdie stamme word getoon om as aanvangskulture gebruik te word vir AMG, om sodoende die groei van natuurlike melksuur bakterieë te inhibeer. Die teenwoordigheid van die plantarisien peptied in die wyn moet egter nog bewys word. Daar sal ook vasgestel moet word of die peptied biologies aktief is en nie deur wynverwante faktore geïnhibeer word nie.

Bacteriocinas produzidas por bactérias láticas (BAL) apresentam um importante potencial de aplicação na bioconservação de alimentos, por sua ação antimicrobiana contra algumas espécies de microrganismos patogênicos de relevância, como Listeria monocytogenes. Este estudo analisou o efeito da interação entre cepas selecionadas de BAL produtoras de bacteriocinas com outras BAL viáveis ou não viáveis (bacteriocinogênicas ou não) na indução da produção de bacteriocinas. O efeito dos metabólitos produzidos por estas cepas na indução da bacteriocinogênese também foi avaliado. As cepas produtoras de bacteriocinas selecionadas para o estudo foram Lactobacillus sakei MBSa1, produtora de sakacina A e Pediococcus acidilactici ET34, produtora de pediocina, isoladas de salame e salmão defumado, respectivamente. A produção de pediocina por P. acidilactici ET34 foi avaliada também em leite em pó desnatado reconstituído, além de meio de cultura (caldo MRS). Os resultados indicaram que, quando em co-cultura com Enterococcus faecalis ATCC12755, Lactobacillus sakei ATCC15521 ou Listeria monocytogenes (cepas 104, 711 e 637), ou na presença do sobrenadante livre de células (SLC) dessas culturas, nenhuma das duas cepas testadas produziu maior quantidade de bacteriocina do que a produzida quando em monocultura ou na ausência do SLC. A bacteriocina produzida por P. acidilactici ET34 apresentou um efeito bacteriostático contra L. monocytogenes 104 no leite em pó desnatado reconstituído nas 12 h analisadas, com extensão da fase lag, de forma dose-dependente. Os resultados indicaram, também, que P. acidilactici ET34 não foi capaz de produzir pediocina no leite em pó desnatado reconstituído quando em monocultura ou em co-cultura, ao contrário do observado para o caldo MRS. Mais investigação é necessária para esclarecer os efeitos de possíveis interações entre as BAL presentes em um alimento, bem como o efeito dos componentes dos alimentos na produção das bacteriocinas pelas BAL bacteriocinogênicas. / Bacteriocins produced by lactic acid bacteria (LAB) present an important application potential in food biopreservation, by their antimicrobial activity against some species of pathogenic microorganisms of relevance, such as Listeria monocytogenes. This study analyzed the effect of the interaction between selected strains of bacteriocin-producing LAB with other viable or non-viable LAB (bacteriocinogenic or not) in the induction of bacteriocin production. The effect of the metabolites produced by these strains on the induction of bacteriocinogenesis was also evaluated. The bacteriocin-producing strains selected for the study were Lactobacillus sakei MBSa1, producer of sakacin A and Pediococcus acidilactici ET34, producer of pediocin, isolated from salami and smoked salmon, respectively. The production of pediocin by P. acidilactici ET34 was also evaluated in reconstituted skimmed milk powder as well as culture medium (MRS broth). The results indicated that when co-cultivated with Enterococcus faecalis ATCC12755, Lactobacillus sakei ATCC15521 or Listeria monocytogenes (strains 104, 711 and 637), or in the presence of the cell free supernatant (SLC) of these cultures, neither of the two strains tested produced greater amount of bacteriocin than that produced in monoculture or in the absence of SLC. The bacteriocin produced by P. acidilactici ET34 presented a bacteriostatic effect against L. monocytogenes 104 in skimmed milk powder reconstituted in 12h, with extension of lag phase, in a dose-dependent manner. The results also indicated that P. acidilactici ET34 was not able to produce pediocin in the reconstituted skimmed milk powder when in monoculture or in co-culture, unlike that observed for the MRS broth. More research is needed to clarify the effects of possible interactions between BAL present in a food and the effect of food components on bacteriocin production by bacteriocinogenic BAL.

A bactéria Lactococcus lactis subsp. lactis CECT-4434 foi empregada para investigar o efeito da composição do meio de cultivo na produção biotecnológica de biossurfactante e, adicionalmente bacteriocina. Utilizou-se resíduos agroindustriais, tais como soro de leite e vinhaça de uva, para formular meios de cultivos mais econômicos e naturais, suplementados sacarose e extrato de levedura. Um planejamento fatorial fracionado 24, com adição de três ensaios nos pontos centrais foi empregado para avaliar a influência destas variáveis. A produção de biossurfactante foi influenciada positivamente pela concentração soro de leite, onde 15 % deste demonstrou melhor resultado reduzindo a tensão superficial em cerca de 18,1 mN/m, alcançando produção máxima de biossurfactante equivalente em surfactina de 11,02 mg/L. Em relação à síntese de bacteriocina, a fonte de carbono adicional (sacarose) interferiu de forma antagonista, ou seja, quanto menor a concentração de sacarose, maior a síntese de bacteriocina (com aumento da zona de inibição em 14,2% contra Staphylococcus aureus CECT-239). Observou-se que o ensaio conduzido em biorreator, sob microaeração com 5% de oxigênio dissolvido, promoveu maior produção de biossurfactante (11,6 mg/L) quando comparados aos estudos conduzidos com maior concentração de oxigênio entre 30 a 100%, com produção em média de 2,3 mg/mL. Destaca-se que nenhum estudo da influência do oxigênio dissolvido, principalmente em microaerofilia, para a produção de biossurfactante por bactérias láticas já havia sido realizado. Ademais, o biossurfactante produzido se mostrou altamente estável frente a valores extremos de pH e temperatura, além de demonstrar notável propriedade antimicrobiana e antiadesiva, inibindo Listeria monocytogenes NADC 2045 e Salmonella entérica<i/> CECT-724 em mais de 90%. / Lactococcus lactis subsp. lactis CECT-4434 was used to investigate the effect of the composition of the culture media on the biotechnological production of biosurfactant and bacteriocin additionally. Agroindustrial residues, such as whey and grape vinasse, were used to formulate more economical and natural culture media, supplemented with sucrose and yeast extract. A fractional factorial design 24, with addition of three runs at the central points was used to evaluate the influence of these variables. The biosurfactant production was positively influenced by the concentration of whey, where 15% showed a better result reducing the surface tension by 18.1 mN/m, reaching a maximum production of biosurfactant equivalent in surfactin of 11.02 mg/L. In relation to bacteriocin synthesis, the sucrose interfered in an antagonistic way, that is, the lower the sucrose concentration, the greater the bacteriocin synthesis (with an increase in the zone of inhibition in 14.2% against Staphylococcus aureus CECT-239). It was observed that the bioreactor conducted under microaeration with 5% dissolved oxygen promoted a higher biosurfactant production (11.6 mg/L) when compared to studies conducted with a higher concentration of oxygen between 30 and 100%, with production on average 2.3 mg/mL. It is noteworthy that no study of the influence of dissolved oxygen, mainly in microaerophilic, for the biosurfactant production by lactic acid bacteria had already been carried out. In addition, the biosurfactant produced proved to be highly stable against extreme values of pH and temperature, and demonstrated remarkable antimicrobial and antiadhesive properties, inhibiting Listeria monocytogenes NADC 2045 and Salmonella entérica CECT-724 in more than 90%.