Bacteriocins are peptides produced by various species of bacteria, especially lactic acid bacteria, which exhibit a large spectrum of action against spoilage bacteria and foodborne pathogens. Successful application of techniques for quantitative or qualitative bacteriocin determination relies not only on the sensitivity of the test-microorganisms, but also on the agar-medium employed. Cell free supernatants are routinely used to preliminary screen for antimicrobial activity of bacteria by means of the agar well diffusion method, but the supernatant may also include other molecules (such as medium components and/or intracellular compounds) accidentally released during cell free supernatant preparation, which may interfere with the assay. Reproducibility of bacteriocin activity against the same test-microorganisms is an important factor to be considered. Unfortunately, no specific information about bioassays standardization to determine bacteriocin activity is available in the literature. In this work, growth inhibition by means of the agar well diffusion assays were carried out on different agar-media showing a strong dependence on the agar-medium used, indicating that the inhibitory effects could also depend on the diffusion of exudates that are included in the cell-free supernatant. The results presented in this communication show that selection of the agar-medium is crucial for the bioassay response.

Os Bacillus sp. produzem péptidos antimicrobianos incluindo bacteriocinas que são péptidos de síntese ribossomal. As bacteriocinas têm originado um grande interesse em várias áreas, desde a saúde humana até à indústria alimentar. Este trabalho teve como objetivo a seleção de 5 isolados de Bacillus sp. do Coleção de Bactérias Esporulantes dos Açores e a otimização das condições de crescimento e a maximização da produção da atividade antimicrobiana em meio liquido de 1 destes isolados, assim como a caracterização e purificação parcial das moléculas ativas. [...].

As bactérias produzem várias substâncias antibacterianas desde antibióticos, lisozimas, exotoxinas e também bacteriocinas. As bacteriocinas são péptidos antimicrobianos de síntese ribossomal que têm suscitado um interesse crescente no combate a estirpes resistentes a antibióticos. Algumas espécies de Bacillus produzem bacteriocinas/BLIS que possuem ação contra MRSA e outras estirpes resistentes. As aplicações das bacteriocinas vão desde a Saúde Humana até à utilização como bioconservantes. O objetivo deste trabalho foi o estudo e caracterização da atividade antibacteriana de 2 isolados de Bacillus (S54c e S150c) da Coleção Açoriana de Bactérias Esporulantes, bem como das suas moléculas ativas. Os dois isolados mostraram maior crescimento e maior atividade antibacteriana em meio Brain Heart Infusion após 18 horas de incubação. Para o sobrenadante filtrado destes isolados e para as frações ativas do sobrenadante do isolado S54c foi determinada a estabilidade térmica e a sensibilidade a enzimas hidrolíticas. O resultado destes testes foi avaliado pela determinação da atividade antibacteriana pelo método de difusão em agar contra a bactéria Micrococcus luteus. A atividade dos isolados S54c e S150C contra Staphylococcus aureus e MRSA foi avaliada pelo método descrito para bactérias esporulantes e o espectro de atividade do sobrenadante do isolado S54c foi determinado. O sobrenadante liofilizado do isolado S54c foi fracionado por cromatografia de exclusão molecular, coluna Sephacryl S-200 40 ml, e as frações positivas visualizadas em SDS-PAGE. Os sobrenadantes dos isolados S54c, S150c e a fração>30kDa do sobrenadante do isolado S54c possuem estabilidade térmica até aos 100ºC, a fração entre os 3-30kDa do isolado S54c é estável até aos 121ºC. Mostrou-se que a atividade antibacteriana dos isolados S54c, S150c e a fração>30kDa (S54c) é hidrolisada pela proteinase K e quimiotripsina, enquanto a fração entre os 3-30 kDa(S54c) apenas é hidrolisada pela proteinase K. Os resultados obtidos indicam que a atividade antibacteriana é de origem proteica e permitem classificar estas moléculas como BLIS. Os isolados S54c e S150c mostraram atividade contra S.aureus e MRSA, contudo o S150c apresentou uma atividade mais reduzida. O sobrenadante filtrado do isolado S54c mostrou atividade contra M.luteus e B.cereus e o fracionamento em cromatografia de exclusão molecular resultou na identificação de uma fração ativa. A análise da sequência parcial do gene 16S mostrou que o isolado S54c é uma estirpe de B.cereus. Este trabalho permitiu identificar parcialmente BLIS produzidas por B.cereus S54c e que podem vir a ser purificadas por técnicas cromatográficas.

Bacteriocin B231 produced by Lactobacillus pentosus, isolated from an artisanal raw cow's milk protected designation of origin Portuguese cheese, is a small protein with an apparent relative mass of about 5 kDa and active against a large number of Listeria monocytogenes wild-type strains, Listeria ivanovii and Listeria innocua. Bacteriocin B231 production is highly dependent on the type of the culture media used for growth of Lact. pentosus B231. Replacement of glucose with maltose yielded the highest bacteriocin production from eight different carbon sources. Similar results were recorded in the presence of combination of glucose and maltose or galactose. Production of bacteriocin B231 reached maximal levels of 800 AU/ml during the stationary phase of growth of Lact. pentosus B231 in MRS broth at 30 °C. Bacteriocin B231 (in cell-free supernatant) was sensitive to treatment with trypsin and proteinase K, but not affected by the thermal treatment in range of 55-121 °C, or freezing (-20 °C). Bacteriocin production and inhibitory spectrum were evaluated. Gene encoding plantaricin S has been detected in the genomic DNA. Virulence potential and safety of Lact. pentosus B231 were assessed by PCR targeted the genes gelE, hyl, asa1, esp, cylA, efaA, ace, vanA, vanB, hdc1, hdc2, tdc and odc. The Lact. pentosus B231 strains harbored plantaricin S gene, while the occurrence of virulence, antibiotic resistance and biogenic amine genes was limited to cytolysin, hyaluronidase, aggregation substance, adhesion of collagen protein, gelatinase, tyrosine decarboxylase and vancomycin B genes.

Lactobacillus plantarum B391, a strain isolated from an artisanal French cheese, is a producer of a bacteriocin, expressing activity against Enterococcus faecalis NCTC 775, Clostridium perfringens NCTC 13170 and several Listeria monocytogenes strains. High stability was recorded after heat treatment at 121 °C for 20 min and when stored at 4 °C for more than 40 days. A challenge test performed in milk for 11 days showed potential for the control of L. monocytogenes. In the presence of the lytic bacteriocin B391, L. monocytogenes cells present numerous morphology modifications of cell shape and surface structure as well as in the cell division pattern, resulting ultimately in lysis. The high level of Listeria growth inhibition obtained in the presence of Lb. plantarum B391, and the stability of B391 bacteriocin for a long period of time, make this strain potentially interesting to use in milk products to increase food safety.

Two methods of purification of the plantaricin ST31, a bacteriocin produced by Lactobacillus plantarum ST31 are used in this study – the method of ammonium sulfate precipitation, Sep-pack C18 cartridge and reverse-phase HPLC chromatography on C18 Nucleosil column, and the method of direct purification by cation exchange SP Sepharose Fast Flow column Amersham (Pharmacia Biotech). The purity of the products from the two experimental protocols are examined for their molecular weight, aminoacid composition and sequence. Comparison of results show that the plantaricins purified with the two methods are identical. Both methods may be used to purify plantaricin ST31. Comparison of the yield in the purification protocols is 0.8% in the HPLC experimental protocol and 5.9% in the cation-exchange chromatography method.

Probiotics are living microorganisms used as nutritional additives that confer health benefits on the host. Their use in food products is very attractive, especially if they could also inhibit important foodborne athogens. In this study, antimicrobial activity against several foodborne pathogens was screened for 280 lactic acid bacteria (LAB) isolated from di erent food products and the probiotic characteristics of bacteriocinogenic isolates were evaluated. Seven out of 280 LAB isolates were selected due to their bacteriocinogenic properties and identified by 16S rRNA gene sequence analysis as Pediococcus pentosaceus (n = 6) and Lactobacillus plantarum (n = 1). Virulence factors and antibiotic resistances were not detected for any of the isolates. Except for L. plantarum R23, all the isolates were able to survive through the simulated gastrointestinal tract conditions. Only P. pentosaceus CFF4 was able to adhere to Caco-2 cells after the simulated gastrointestinal tract passage. In conclusion, even though in vivo studies should be performed, P. pentosaceus CFF4, which was also able to inhibit the growth of foodborne pathogens in vitro, seems to be a potential probiotic to be used in the food industry.

Bacteriocin producing lactic acid bacteria (LAB) cultures can be used as biopreservatives in fermented food products; thus the food industry is interested in stable cultures that produce bacteriocins consistently. Inhibtion of Listeria spp. by bacteriocinogenic Pediococcus acidilactici and Lactobacillus plantarum (both isolated from fermented meats) was investigated under conditions of stress induced by low pH and high salt concentrations. Listeria monocytogenes serogroup IIb (from cheese), L. monocytogenes serogroup IVb (from cheese), L. monocytogenes serogroup IIb (from ground beef) and Listeria innocua NCTC 11288 were used as target strains. P. acidilactici and Lb. plantarum demonstrated antilisterial activity under the stress conditions investigated (pH 3.5; pH 8.5; 7.5% NaCl). However, activity was dependent on the stress conditions applied and on the target organism. L. monocytogenes serogroup IIb (from ground beef) and L. innocua C 11288 were, respectively the most sensitive and the most resistant to the cell-free supernatants produced by the LAB investigated.

Pediococcus pentosaceus SB83 is a bacteriocinogenic culture having potential use as a vaginal probiotic. The objective of this study was to evaluate the behaviour of P. pentosaceus SB83 incorporated in a liquid system for eventually formulating a gel for vaginal delivery. The vaginal probiotic incorporated into glycerol was able to survive in simulated vaginal fluid at normal vaginal pH (4.2) and at pH of vaginal infections (5.5 and 6.5). The probiotic can be stored at 4 °C for a longer period of time than at room temperature, however, after 13 weeks of storage at low temperature, there was a total loss of viable cells. The probiotic strain incorporated into glycerol showed bacteriocinogenic activity in simulated vaginal fluid, although the antimicrobial activity against Listeria monocytogenes declined during storage. This study showed the behaviour of P. pentosaceus SB83 in glycerol and in simulated vaginal fluid. However, it is necessary to optimise the formulation to produce an actively probiotic vaginal gel.

This study was conducted to evaluate the ability of Pediococcus acidilactici HA-6111-2, a PA-1 bacteriocinproducing lactic acid bacterium (LAB), isolated from ‘‘Alheira” to inhibit a cocktail of Listeria innocua strains during production and shelf-life of these products. The bacteriocinogenic culture reduced the Listeria population below the detection limit (1.5log CFU/g) and had no effect on the growth of the natural LAB flora or on the pH. Pathogenic organisms were not detected in any sample. The presence of some virulence factors and antibiotic resistances of the strain to be used as a bioprotective culture were investigated. P. acidilactici HA-6111-2 did not produce any of the biogenic amines tested; no formation of biofilms was observed; more L(+)lactic acid was produced than its isomer D(-); no gelatinase, DNase or lipase activity was recorded; no structural genes for the haemolysin, enterococcal surface protein, hydrolytic compounds, aggregation protein and cell-wall adhesins were detected, no significant antibiotic resistances were found. P. acidilactici HA-6111-2 appears to have potential as a bioprotective culture during ‘‘Alheira” fermentation. Moreover, a trained panel considered the protected product to be sensorially acceptable.