Heterologous expression of pediocin PA-1 in Pichia pastoris: cloning, expression, characterization, and application in pork bologna preservation.

OBJECTIVE: Pediocin PA-1, an antimicrobial peptide derived from Pediococcus acidilactici PAC1.0, has a potential application as a food preservative thanks to its strong inhibitory activity against the foodborne pathogen L. monocytogenes. This study aimed to produce Pediocin PA-1 from the yeast P. pastoris and evaluate its characteristics. METHODS: Gene encoding Pediocin PA-1 was integrated into P. pastoris X33 genome to establish the strain X33::ped, which could produce and secrete this peptide into culture medium. The antimicrobial activity of Pediocin PA-1 was examined using agar diffusion assay. The stability of pediocin PA-1 was determined based on its remaining antibacterial activity after exposure to proteases and extreme pH and temperatures. The potential use of this bacteriocin in food preservation was demonstrated using the L. monocytogenes infected pork bologna. The anticancer activity of Pediocin PA-1 was also investigated on some cancer cells using MTT assay. RESULTS: We established the yeast P. pastoris X33::ped capable of producing pediocin PA-1 with antimicrobial activity against L. monocytogenes and some other harmful bacteria. Pediocin PA-1 was stable at 100˚C and resistant against pH 1-12 for 1 h, but susceptible to trypsin, α-chymotrypsin, and proteinase K. This peptide could reduce the number of L. monocytogenes in pork bologna by 3.59 log CFU/g after 7 days of storage at 4˚C. Finally, Pediocin PA-1 (25 µg/ml) inhibited the proliferation of A549 and Hela cancer cells. CONCLUSION: We succeeded in producing active Pediocin PA-1 from P. pastoris and demonstrated its potential use in food preservation and pharmaceutical industry.

An oxidation resistant pediocin PA-1 derivative and penocin A display effective anti-Listeria activity in a model human gut environment.

Pediocin PA-1 is a class IIa bacteriocin that is particularly effective against the foodborne pathogen Listeria monocytogenes. The loss of activity of PA-1 pediocin due to methionine oxidation is one of the challenges that limit the wider application of the bacteriocin. In this study, we heterologously expressed an oxidation resistant form of pediocin PA-1, i.e., pediocin M31L, and compared its activity to that of native pediocin PA-1 and to penocin A, a pediocin-like bacteriocin that displays a narrower antimicrobial spectrum. Minimal inhibitory concentration assays revealed that pediocin M31L was as effective as PA-1 and more effective than synthetic penocin A against Listeria with negligible activity against a range of obligate anaerobic commensal gut bacterial species. The anti-Listeria activity of these pediocins was also assessed in a simulated human distal colon model assay using the L. monocytogenes, spiked at 6.5 ± 0.13 Log CFU/mL, as a bioindicator. At 24 h, pediocin M31L and penocin A (2.6 µM) reduced Listeria counts to 3.5 ± 0.4 and 3.64 ± 0.62 Log CFU/mL, respectively, whereas Listeria counts were considerably higher, i.e. 7.75 ± 0.43 Log CFU/mL, in the non-bacteriocin-containing control. Ultimately, it was established that synthetic penocin A and the stable pediocin M31L derivative, heterologously produced, display effective anti-Listeria activity in a human gut environment.

Functional and technological characterization of lactic acid bacteria isolated from Turkish dry-fermented sausage (sucuk).

In this study, 10 lactic acid bacteria were isolated from Turkish fermented sausage (sucuk) and identified as 5 Lactobacillus plantarum, 1 Pediococcus acidilactici, 1 Weissella hellenica, 1 Lactobacillus pentosus, and 2 Lactobacillus sakei. PCR screening of genes encoding plantaricin A and pediocin showed the presence of plantaricin A gene in 9 and pediocin gene in 3 of strains. All isolates showed antibacterial and antifungal effect on most of the tested microorganisms. gad gene, encoding glutamic acid decarboxylase enzyme, was detected in all isolates except Weisella hellenica KS-24. Eight of isolates were determined as gamma-amino butyric acid (GABA) producer in the presence of 53 mM mono sodium glutamate (MSG) by HPLC and TLC analysis. DPPH scavenging activity was observed for all isolates. Additionally, isolates were able to produce exopolysaccharide in the presence of sucrose. The best exopolysaccharide (EPS) production was achieved with L. plantarum KS-11 and L. pentosus KS-27. As a result, this study characterized some techno-functional properties of LAB isolates from sucuk. It was concluded that the isolates studied have the potential to be used in obtaining functional products in meat industry, as well as strain selection may be effective in providing the desired properties in the product.

Pediocin PA-1 production by Pediococcus pentosaceus ET34 using non-detoxified hemicellulose hydrolysate obtained from hydrothermal pretreatment of sugarcane bagasse.

Listeria monocytogenes is one of the foodborne pathogens of most concern for food safety. To limit its presence in foods, bacteriocins have been proposed as natural bio-preservatives. Herein, a bacteriocin was produced on hemicellulose hydrolysate of sugarcane bagasse by Pediococcus pentosaceous ET34, whose genome sequencing revealed an operon with 100% similarity to that of pediocin PA-1. ET34 grown on hydrolysate-containing medium led to an increase in the expression of PA-1 genes and a non-optimized purification step sequence resulted in a yield of 0.8 mg·L-1 of pure pediocin (purity > 95%). Culture conditions were optimized according to a central composite design using temperature and hydrolysate % as independent variables and validated in 3-L Erlenmeyers. Finally, a process for scaled-up implementation by sugar-ethanol industry was proposed, considering green chemistry and biorefinery concepts. This work stands up as an approach addressing a future proper sugarcane bagasse valorisation for pediocin production.

The case for class II bacteriocins: A biophysical approach using "suicide probes" in receptor-free hosts to study their mechanism of action.

Class II bacteriocins are unmodified membrane-active peptides that act over a narrow spectrum of target bacteria. They bind a specific receptor protein on the membrane to form a pore, leading to membrane permeabilization and cell death. However, little is known about the molecular events triggering the pore formation after the bacteriocin recognizes the receptor. It is not clear yet if the pore is the same receptor forced into an open conformation or if the pore results from the bacteriocin insertion and oligomeric assembly in the lipid bilayer. In order to reveal which model is more suitable to explain the toxicity mechanism, in this work we use chimeric peptides, resulting from the fusion of the bitopic membrane protein EtpM with different class II bacteriocins: enterocin CRL35, pediocin PA-1 and microcin V. E. coli strains lacking the specific receptors for these bacteriocins were chosen as expression hosts. As these constructs display a lethal effect when they are heterologously expressed, they are called "suicide probes". The results suggest that, indeed, the specific receptor would act as a docking molecule more than as a structural piece of the pore, as long as the bacteriocin is somehow anchored to the membrane. These set of chimeric peptides also represent an in vivo system that allows to study the interaction of the bacteriocins with real bacterial membranes, instead of model membranes. Hence, the effects of these suicide probes in membrane fluidity and transmembrane potential were also assessed, using fluorescence spectroscopy. The data show that the different suicide probes are able to increase phospholipid order and depolarize the membranes of receptor-free bacterial cells.

Bioprodução de pediocina por Pediococcus pentosaceus ET34 em resíduo agroindustrial hidrolisado bagaço de cana-de-açúcar / Bioproduction of Pediocin by Pediococcus pentosaceus ET34 in hydrolyzed sugar cane bagasse agroindustrial residue

Um dos grandes desafios nas indústrias alimentícia, farmacêutica e agropecuária é a busca por novos compostos para substituir os antibióticos. Como possíveis candidatos estão as bacteriocinas para serem utilizados paralelamente aos antibióticos ou até substituí-los. As bactérias ácido-láticas podem produzir substâncias inibitórias semelhantes às bacteriocinas (BLIS - bacteriocin-like inhibitory substances) que possuem efeito bacteriostático ou bactericida contra diferentes grupos de bactéria sendo largamente utilizadas como bioconservantes alimentares. Neste contexto, o objetivo deste trabalho foi o emprego de um resíduo agroindustrial, o hidrolisado de bagaço de cana-de-açúcar, como fonte de carbono em cultivos fermentativos para produção do BLIS pela cepa Pediococcus pentosaceus ET 34. Resultados revelaram que as células de ET34 foram capazes de crescer utilizando este resíduo agroindustrial como fonte de carbono e ensaios utilizando planejamento fatorial demonstraram que a agitação não influencia na produção de BLIS. Ao escalonar o cultivo em biorreatores, foi verificado que tanto o crescimento como a atividade antimicrobiana foram semelhantes aos obtidos em bancada com exceção da condição de 25% (v/v) de HBC (hidrolisado de bagaço de cana) que devido a maior viscosidade do meio, resultou em uma diminuição no crescimento e de produção de BLIS. O BLIS produzido por células de ET34 utilizando o HBC como fonte de carbono foi parcialmente purificado por sulfato de amônio e demonstrou atividade contra Listeria monocytogenes ATCC 934 e Salmonella enterica CECT 724. Desta maneira, pode-se concluir que Pediococcus pentosaceus ET34 foi capaz de crescer em meio contendo HBC como fonte de carbono produzindo BLIS em seu sobrenadante com ação frente a diferentes bactérias patogênicas. A possibilidade de utilizar uma fonte alternativa de carbono pode diminuir o custo de processo consideravelmente. Além disso, ensaios de planejamento fatorial, superfície de resposta e escalonamento em biorreator de bancada indicaram que concentrações baixas de HBC (5-15%, v/v) a 35 °C resultaram na maior produção de BLIS

The great challenge in the food, pharmaceutical and agricultural industries is the search for new compounds to replace antibiotics. Bacteriocins are possible candidates that can be used in parallel with the antibiotics or even to replace them. Lactic-acid bacteria can produce bacteriocin inhibitory substances (BLIS - bacteriocin-like inhibitory substances) that have a bacteriostatic or bactericidal effect against different bacterial species and are widely used as food bioconservatives. In this context, the aim of this work was to use of an agroindustrial waste, hydrolyzed sugarcane bagasse, as a carbon source in fermentative cultures for the production of BLIS by Pediococcus pentosaceus ET 34 strain. Results revealed that ET34 cells were able to grow using this agroindustrial residue as a carbon source, and trials using factorial design showed that agitation did not influence on the production of BLIS. When it was perform cultivation scale up in bioreactors, it was verified that both the growth and the antimicrobial activity were similar to those obtained in the workbench with the exception of the condition of 25% (v/v) of HBC (sugarcane bagasse hydrolyzate) that due to its higher viscosity, resulted in a decrease in growth and BLIS production. BLIS produced by ET34 cells using HBC as a carbon source that was partially purified by ammonium sulfate showed activity against Listeria monocytogenes ATCC 934 and Salmonella enterica CECT 724. Thus, it can be concluded that Pediococcus pentosaceus ET34 was able to grow in medium containing HBC as carbon source producing BLIS in its supernatant with action against different pathogenic bacteria. The possibility of using an alternative carbon source can greatly reduce the process cost. In addition, factorial design, response surface and scale up trials in bench bioreactors indicated that low concentrations of HBC (5-15% v/v) at 35 ºC resulted in higher BLIS production

Papel da interação entre bactérias láticas isoladas de alimentos na produção de bacteriocinas / Role of interactions among lactic acid bacteria isolated from foods on production of bacteriocins

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

Pediocin-like bacteriocins: new perspectives on mechanism of action and immunity.

This review attempts to analyze the mechanism of action and immunity of class IIa bacteriocins. These peptides are promising alternative food preservatives and they have a great potential application in medical sciences. Class IIa bacteriocins act on the cytoplasmic membrane of Gram-positive cells dissipating the transmembrane electrical potential by forming pores. However, their toxicity and immunity mechanism remains elusive. Here we discuss the role of the mannose phosphotransferase system (man-PTS) as the receptor for class IIa bacteriocins and the influence of the membrane composition on the activity of these antimicrobial peptides. A model that is consistent with experimental results obtained by different researchers involves the non-specific binding of the bacteriocin to the negatively charged membrane of target bacteria. This step would facilitate a specific binding to the receptor protein, altering its functionality and forming an independent pore in which the bacteriocin is inserted in the membrane. An immunity protein could specifically recognize and block the pore. Bacteriocins function in bacterial ecosystems and energetic costs associated with their production are also discussed. Theoretical models based on solid experimental evidence are vital to understand bacteriocins mechanism of action and to promote new technological developments.

Pediococcus spp.: An important genus of lactic acid bacteria and pediocin producers.

Probiotics have gained increasing attention due to several health benefits related to the human digestive and immune systems. Pediococcus spp. are lactic acid bacteria (LAB) that are widely described as probiotics and characterized as coccus-shaped bacteria (arranged in tetrads), Gram-positive, non-motile, non-spore forming, catalase-negative, and facultative anaerobes. There are many Pediococcus strains that produce pediocin, an effective antilisterial bacteriocin. Pediocins are small, cationic molecules consisting of a conserved hydrophilic N-terminal portion containing the YGNGV motif and an amphiphilic or hydrophobic C-terminal variable portion. A number of studies have been developed with Pediococcus isolated from multiple biological niches to conduct fermentation processes for pediocin or Pediococcus cell production. This review gathers the most significant information about the cultivation, mode of action, and variability of bacteriocins produced by Pediococcus spp., emphasizing their applications in the areas of food and clinical practice. This updated panorama assists in delimiting the challenges that still need to be overcome for pediocin use to be approved for human consumption and the food industry.