Análise in vitro da ação antimicrobiana e antioxidante de bactérias láticas potencialmente probióticas

A microbiota é definida como um conjunto de microrganismos que co-existem em determinado local. Alterações em sua composição pode provocar diversas desordens de seu funcionamento normal. O uso dos probióticos, bactérias vivas que podem beneficiar o hospedeiro quando administradas em concentrações adequadas, vem se popularizando devido aos seus benefícios para a saúde animal através de diferentes ações como a exclusão competitiva e atividade antimicrobiana. Assim, o presente trabalho tem como objetivo analisar a capacidade antimicrobiana e antioxidante de 5 linhagens probióticas.info:eu-repo/semantics/publishedVersio

Avaliação in vitro dos potenciais antibacteriano e antioxidante de paraprobióticos de diferentes linhagens de lactobacilos

Paraprobióticos são frações inativadas de bactérias probióticas que também são capazes de desempenhar efeitos benéficos ao organismo hospedeiro. Destacam-se pela segurança para pacientes com alterações do sistema imunológico, tendo em vista que as bactérias apresentam-se inativadas. Além disso, apresentam potencial antioxidante, sendo capazes de combater radicais livres, produzidos em excesso em processos infecciosos, inclusive nos mediados por bactérias como Staphylococcus aureus e Pseudomonas aeruginosa, espécies patogênicas oportunistas, com resistência a antibióticos e que estão associadas a infecções em diferentes tecidos, em especial à pele, potencialmente comprometendo a cicatrização.info:eu-repo/semantics/publishedVersio

Bioprospecting of the probiotic potential of yeasts isolated from a wine environment

Autochthonous yeasts of oenological origin are adapted to highly stressful and selective environments, which makes them potential candidates for probiotics. The objective of the present study was to explore the probiotic potential of 96 native yeasts of oenological origin, their biosafety, resistance to gastrointestinal tract conditions and adhesion properties. Regarding biosafety, 66 isolates shown negative hemolytic activity, negative urease activity and susceptibility to 3 or more antifungals. After the gastrointestinal resistance test, 15 isolates were selected that showed growth at different temperatures, tolerance to low pH and the presence of bile salts in in vitro tests. In general, survival after simulated conditions of the gastrointestinal tract was high and more restrictive was the duodenal. The results of the adhesion properties showed highly variable hydrophobicity and a high percentage of autoaggregation at 24 h. The maximum production of biofilm was detected in the Pichia strains. Of a total of 96 yeast strains, 15 non-Saccharomyces yeasts presented suitable properties as probiotic candidates. The native winemaking strains performed better than the reference probiotic strain, Saccharomyces cerevisiae var. boulardii CNCM I-745, which reaffirms that these strains are promising probiotic candidates and further studies are necessary to confirm their probiosis.Fil: Vergara Alvarez, Silvia Cristina. Universidad Nacional de San Juan. Facultad de Ingeniería. Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - San Juan; ArgentinaFil: Leiva Alaniz, María José. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - San Juan; Argentina. Universidad Nacional de San Juan. Facultad de Ingeniería. Instituto de Biotecnología; ArgentinaFil: Mestre Furlani, María Victoria. Universidad Nacional de San Juan. Facultad de Ingeniería. Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - San Juan; ArgentinaFil: Vazquez, Fabio. Universidad Nacional de San Juan. Facultad de Ingeniería. Instituto de Biotecnología; ArgentinaFil: Mancha Agresti, Pamela. Centro Federal de Educação Tecnológica; BrasilFil: Cristina Nally, María. Universidad Nacional de San Juan. Facultad de Ingeniería. Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - San Juan; ArgentinaFil: Maturano, Yolanda Paola. Universidad Nacional de San Juan. Facultad de Ingeniería. Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - San Juan; Argentin

Recombinant Probiotics and Microbiota Modulation as a Good Therapy for Diseases Related to the GIT

Many diseases that affect the gastrointestinal tract (GIT) have great influence on the quality of life of the majority of patients. Many probiotic strains are being highly studied as a promising candidate due to their beneficial effect reported in the GIT. With the purpose of increasing the beneficial characteristics of some probiotics strains and, consequently, to improve further the reported results, many probiotic strains expressing or encoding different proteins, with anti-inflammatory activities, have been developed. These recombinant strains have been reported as good candidates for the treatment of different pathological conditions, especially colitis and mucositis disease since they have been shown to have positive results and good perspectives for GIT inflammation. Thus, this chapter will first address the aspects of the gastrointestinal tract in humans as well as its microbiota. In a second moment, it will discuss about chronic diseases, mainly the intestinal ones. Finally, it will discuss about probiotics, especially concerning on lactic acid bacteria (LAB), and its action in the prevention and treatment of these diseases. At the final part, we will point out aspects on the development of recombinant strains and the results found in the literature on disease models

Vector Development Timeline for Mucosal Vaccination and Treatment of Disease Using Lactococcus lactis and Design Approaches of Next Generation Food Grade Plasmids

Lactococcus lactis has been used historically in fermentation and food preservation processes as it is considered safe for human consumption (GRAS—Generally Recognized As Safe). Nowadays, in addition to its wide use in the food industry, L. lactis has been used as a bioreactor for the production of molecules of medical interest, as well as vectors for DNA delivery. These applications are possible due to the development of promising genetic tools over the past few decades, such as gene expression, protein targeting systems, and vaccine plasmids. Thus, this review presents some of these genetic tools and their evolution, which allow us to envision new biotechnological and therapeutic uses of L. lactis. Constitutive and inductive expression systems will be discussed, many of which have been used successfully for heterologous production of different proteins, tested on animal models. In addition, advances in the construction of new plasmids to be used as potential DNA vaccines, delivered by this microorganism, will also be viewed. Finally, we will focus on the scene of gene expression systems known as “food-grade systems” based on inducing compounds and safe selection markers, which eliminate the need for the use of compounds harmful to humans or animal health and potential future prospects for their applications

Microencapsulation of Lactic Acid Bacteria Improves the Gastrointestinal Delivery and in situ Expression of Recombinant Fluorescent Protein

The microencapsulation process of bacteria has been used for many years, mainly in the food industry and, among the different matrixes used, sodium alginate stands out. This matrix forms a protective wall around the encapsulated bacterial culture, increasing its viability and protecting against environmental adversities, such as low pH, for example. The aim of the present study was to evaluate both in vitro and in vivo, the capacity of the encapsulation process to maintain viable lactic acid bacteria (LAB) strains for a longer period of time and to verify if they are able to reach further regions of mouse intestine. For this purpose, a recombinant strain of LAB (L. lactis ssp. cremoris MG1363) carrying the pExu vector encoding the fluorescence protein mCherry [L. lactis MG1363 (pExu:mCherry)] was constructed. The pExu was designed by our group and acts as a vector for DNA vaccines, enabling the host cell to produce the protein of interest. The functionality of the pExu:mCherry vector, was demonstrated in vitro by fluorescence microscopy and flow cytometry after transfection of eukaryotic cells. After this confirmation, the recombinant strain was submitted to encapsulation protocol with sodium alginate (1%). Non-encapsulated, as well as encapsulated strains were orally administered to C57BL/6 mice and the expression of mCherry protein was evaluated at different times (0–168 h) in different bowel portions. Confocal microscopy showed that the expression of mCherry was higher in animals who received the encapsulated strain in all portions of intestine analyzed. These results were confirmed by qRT-PCR assay. Therefore, this is the first study comparing encapsulated and non-encapsulated L. lactis bacteria for mucosal DNA delivery applications. Our results showed that the microencapsulation process is an effective method to improve DNA delivery, ensuring a greater number of viable bacteria are able to reach different sections of the bowel

Growth differentiation factor 11 delivered by dairy Lactococcus lactis strains modulates inflammation and prevents mucosal damage in a mice model of intestinal mucositis

Mucositis is an inflammation of the gastrointestinal mucosa that debilitate the quality of life of patients undergoing chemotherapy treatments. In this context, antineoplastic drugs, such as 5-fluorouracil, provokes ulcerations in the intestinal mucosa that lead to the secretion of pro-inflammatory cytokines by activating the NF-κB pathway. Alternative approaches to treat the disease using probiotic strains show promising results, and thereafter, treatments that target the site of inflammation could be further explored. Recently, studies reported that the protein GDF11 has an anti-inflammatory role in several diseases, including in vitro and in vivo results in different experimental models. Hence, this study evaluated the anti-inflammatory effect of GDF11 delivered by Lactococcus lactis strains NCDO2118 and MG1363 in a murine model of intestinal mucositis induced by 5-FU. Our results showed that mice treated with the recombinant lactococci strains presented improved histopathological scores of intestinal damage and a reduction of goblet cell degeneration in the mucosa. It was also observed a significant reduction of neutrophil infiltration in the tissue in comparison to positive control group. Moreover, we observed immunomodulation of inflammatory markers Nfkb1, Nlrp3, Tnf, and upregulation of Il10 in mRNA expression levels in groups treated with recombinant strains that help to partially explain the ameliorative effect in the mucosa. Therefore, the results found in this study suggest that the use of recombinant L. lactis (pExu:gdf11) could offer a potential gene therapy for intestinal mucositis induced by 5-FU

Utilização de uma linhagem invasiva de Lactococcus lactis como veículo para a entrega de um plasmídeo vacinal codificando o antígeno Ag85A de Mycobacterium tuberculosis em células mamíferas e avaliação do perfil de resposta imunológica gerado em modelo murino

Exportado OPUSMade available in DSpace on 2019-08-13T18:32:44Z (GMT). No. of bitstreams: 1 tese_final_060814_pdf.pdf: 2523945 bytes, checksum: 412a64ce78884b17d17eca22843d6084 (MD5) Previous issue date: 14O uso de bactérias como veículos para a entrega de plasmídeos vacinais por via das mucosas constitui uma estratégia de vacinação promissora. Neste contexto, bactérias patogênicas atenuadas como Shigella, Yersinia, Listeria e Salmonella vêm sendo utilizadas para a entrega de plasmídeos vacinais às células mamíferas, embora apresentem risco de reversão ao seu fenótipo patogênico. Visando contornar este problema, a utilização de bactérias não patogênicas, tais como as Bactérias Lácticas (BL), vem sendo proposta. Dentro do grupo das BL, Lactococcus lactis vem sendo extensivamente utilizado para a produção e entrega de antígenos e citocinas em nível de mucosas. Estudos envolvendo esta bactéria têm também focado em sua utiilização como veículo para entrega de vacinas gênicas, sendo desenvolvidas linhagens invasivas de L. lactis para aumentar a eficiência de entrega das referidas vacinas às células do hospedeiro. Nosso grupo de pesquisa desenvolveu um plasmídeo replicativo em L. lactis, contendo um cassete de expressão eucariótica, o pValac (Vaccination usig lactic acid bacteria). Assim, o uso de linhagens invasivas de L. lactis para a entrega do vetor pValac expressando o antígeno Ag85A de M. tuberculosis, poderia representar uma nova estratégia para o controle da tuberculose. Assim sendo, o objetivo deste trabalho foi a construção do plasmídeo vacinal pValac:Ag85A e a verificação de sua funcionalidade in vitro, utilizando células eucarióticas da linhagem CHO (Chinese Hamster Ovary), bem como sua clonagem na linhagem invasiva L. lactis FnBPA+, gerando assim, a linhagem L. lactis FnBPA+ (pValac:Ag85A). Esta linhagem, por sua vez, foi utilizada em experimentos de imunização de camundongos C57BL/6 e as respostas imunes humoral e celular, geradas pela mesma, foram avaliadas. Como um todo, observou-se uma polarização da resposta imune para o padrão Th1, nos animais imunizados com a referida linhagem em relação aos animais dos grupos controles. Além disso, observou-se a produção das imunoglobulinas IgG e sIgA nas mucosas, especificamente na mucosa respiratória. Em suma, este projeto constitui um primeiro passo rumo à validação da eficácia e efetividade de novas vacinas gênicas baseadas em bactérias lácticas geneticamente modificadas, por via de administração em mucosas.Using bacteria as a vehicle for orally delivering vaccines plasmids is considered a promissing vaccination strategy. Thus, attenuated pathogenic bacteria, such as Shigella, Yersinia, Listeria and Salmonella are being used for delivering plasmids of vaccine type to mammal cells. However, there is a risk of reversing to its wild phenotype. In order to counteract this problem, we propose the use of non pathogenic bacteria, as lactic bacteria (BL). Within the group of lactic bacteria, the Lactococcus lactis is deemed as a model microorganism, which is being extensively used for antigen and cytokines production and delivery to the mucosal level. Recently studies about this bacteria have focused on their usage as vehicles for the delivery of genic vaccines. For this, invasive strains of L. lactis have been developed in order to increase the delivery efficiency of these vaccines to host cells. Furthermore, our investigation team developed a plasmid that replicates in L. lactis and contains a eukaryotic expression cassette named pValac (Vaccination using lactic acid bactéria). In this way, the use of invasive strains of L. lactis for the delivery of vector pValac expressing the antigen Ag85A of Mycobacterium tuberculosis could represent a new strategy for controlling tuberculose. The aim of this investigation was not only the development of plasmid vaccine pValac:Ag85A and its functional evaluation in vitro but also its clonation in its L. lactis FnBPA+ invasive strain for using this system as a potential genic vaccine. The highly functional performance of the eukaryotic expression cassette when coding antigen Ag85A was confirmed through the detection of Ag85A protein through of immunocytochemistry; and flow cytometry after tansfecting pValac within mammalian cells. Finally, plasmid pValac:Ag85A was transformed into invasive L. lactis strain, originating L. lactis FnBPA+ (pValac:Ag85A) strain. This strain was used in C57BL/6 mice intranasal immunization. The humoral and cellular immune responses were evaluated. We observed a polarization of the immune response towards a Th1, in immunized mice with it strain. In the respiratory mucosal, we detected production of both, IgG and sIgA, antibody. In this way, this project is the first step towards the effective validation of new genetic vaccines based on lactic bacteria genetically modified administered in mucosal level

New Approaches for the design of DNA vaccine vectors delivered by lactic acid bacteria

Lactic Acid Bacteria (LAB) have been bioengineered for mucosal delivery of prophylactic, pro-inflammatory and useful molecules that include bioactive peptides, cytokines, enzymes, allergens and DNA. One of the many benefits that have been attributed to these has been the modulation of immune responses. Regarding LAB as a means of delivery, it is important to highlight that this bacteria have the ability to resist gastric and bile juices allowing their survival and transit through the gastrointestinal tract [1, 2] and conferring them the capability to delivery molecules directly at the intestinal mucosal surface. The Food and Drug Administration (FDA) of the U.S.A. consider Lactobacilli and Lactococcus lactis safe or "GRAS" (Generally Recognized As Safe), and according to with European Food Safety Authority (EFSA), these species have fulfilled the criteria of the competent Qualified Presumption of Safety (QPS) status. Increase knowledge in genetic engineering technologies have made it possible to incorporating new genes, as well as modifying the metabolic functions of these important microorganisms which has lead to improvements in health and food technology [3, 4].The oral administration of live recombinant microorganisms such as LAB can be considered as bio-drugs or bio-pharmaceuticals. This strategy can be used not only in the prevention or in the treatment of different diseases, but also in the development of innovative drugs and vaccines [5, 6]. DNA vaccines are constructions based on plasmids that combine sequences that allows its replication in different bacterial species (including E. coli and LAB) with others that are necessary to express the transgene of interest in vertebrate cells after delivery (Williams, 2013). These kind of vaccines using LAB as delivery vehicles are extremely safe and because they cannot revert to a disease causing form (as occurs with viral vector) since they only encode and express the target antigens and not virulence factors (Premenko-Lanier et al, 2004).Only a handful of groups are performing DNA vaccine design vectors that can be delivered by LAB. One is being led by Dr. Desai; who recently published the construction of the pPERDBY plasmid, in which the reporter gene is in the backbone of the plasmid allowing the cloning of the gene of interest in frame with a reporter gene [11]. Our group, led by Dr. Azevedo developed in 2009, the pValac vector, the first vector designed for DNA vaccines to be delivered by LAB [12] Recently we have published a now DNA vaccine vector called pExu [13]. The pExu vector has some attractive characteristics, such as containing the theta origin replication which offers a higher structural and segregational stability, and has showed exceptional results in in vivo test [13], and can also stably maintain large heterologous DNA inserts [14, 15]. The most common promoter and polyA signals used in DNA vaccine vectors are the constitutive human Cytomegalovirus (CMV) promoter and the bovine growth hormone gene because these increase transcription of mRNA that improves transgene expression (Williams, 2013).All of these plasmids are only being used in ?proof- of- concept? studies since these vectors having antibiotic resistance markers that for legal and ethical reasons are not acceptable which might compromise their applications in health treatments. For genetically modified LAB to be used, the development of food-grade cloning systems and biological containment systems are part of the solution for these to be approved for use in human trials.Johansen (1999) defined food-grade recombinant microorganism as those that contain DNA from the same genus or from other GRAS microorganisms. Some food-grade vectors have been constructed for LAB, but only for the production of heterologous proteins (Henrich et al., 2002, Leenhouts et al 1998; Simoes-Barbosa et al, 2004), not to deliver foreign DNA. The design of these vectors is based on the replacement of antibiotics resistance selection markers; however, other properties need to be considered in order to construct effective food-grade vectors to be used as DNA vaccines. The size, the number of copies of the plasmid, the ability to correctly translate the protein of interest in host cell, as well as their low capacity of recombination with host?s DNA are some characteristics that need to be considered. The use DNA delivery vectors by LAB does not require complex technologies and can even replace the industrial production of certain proteinsthat require expensive purification protocols since the host cells would produce the protein of interest for themselves.Fil: Mancha Agresti, Pamela. Universidade Federal de Minas Gerais; BrasilFil: Leblanc, Jean Guy Joseph. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; ArgentinaFil: Azevedo, Vasco. Universidade Federal de Minas Gerais; BrasilFil: Martins Drumond, Mariana. Universidade Federal de Minas Gerais; Brasil. Centro Federal de Educação Tecnológica de Minas Gerais; Brasi