Dissertations / Theses on the topic 'Weissella'

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro Tecnológico, Programa de Pós-Graduação em Engenharia de Alimentos, Florianópolis, 2015.
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A deterioração de alimentos devido à ação de microrganismos é um problema de grande importância que pode gerar grandes perdas econômicas para as indústrias. As bactérias ácido lácticas (BAL) estão entre os principais microrganismos deterioradores de alimentos, e dentre elas, a Weissella viridescens (anteriormente denominada Lactobacillus viridescens) é bastante conhecida por causar deterioração na superfície de carnes e produtos cárneos embalados a vácuo e em diferentes condições de armazenamento, mesmo em condições de refrigeração. A microbiologia preditiva é considerada uma importante ferramenta para descrever o crescimento microbiano em diferentes condições ambientais. Assim sendo, o objetivo geral deste trabalho foi avaliar a capacidade preditiva de um modelo não isotérmico no crescimento de W. viridescens em meio de cultivo, em diferentes perfis de temperatura. Primeiramente o modelo primário de Baranyi e Roberts foi ajustado às curvas de crescimento, anteriormente obtidas no Laboratório de Engenharia Bioquímica (ENGEBIO), de W. viridescens em meio de cultura sob seis diferentes temperaturas isotérmicas (4 a 30 °C). Foram avaliados quatro modelos secundários para descrever a influencia da temperatura sobre os parâmetros de crescimento, que foi melhor descrita pelo modelo da raiz quadrada para o parâmetro µmáx. O modelo secundário para o parâmetro Ymáx foi considerado como a média dos valores obtidos experimentalmente, independente da temperatura, na faixa de temperatura estudada. Com base nos modelos selecionados, o modelo de Baranyi e Roberts (1994) foi utilizado para prever o crescimento de W. viridescens sob condições não isotérmicas. O modelo proposto foi validado com dados experimentais de W. viridescens em meio MRS, sob diferentes perfis de temperatura, envolvendo temperaturas acima da temperatura de refrigeração e simulando oscilações de temperatura em refrigeradores domésticos. Os resultados mostraram que o modelo preditivo estudado tem a possibilidade de ser utilizado para prever a vida útil de produtos cárneos cuja deterioração ocorra, principalmente, em consequência do crescimento de W. viridescens.

Abstract : The food spoilage due to the action of microorganisms is a large problem that can cause economic losses to industries. The lactic acid bacteria (LAB) are one of the main spoilage microorganisms in foods, and among them Weissella viridescens (previously Lactobacillus viridescens) is well known to spoil meat surfaces and vacuum-packed meat products in different storage conditions, even under refrigerated conditions. The predictive microbiology is considered an important tool to describe the microbial behavior under different environmental conditions. Therefore, the aim of this study was evaluate the predictive ability of a non-isothermal model for growth W. viridescens in culture medium, under different temperature profiles. First, the primary model of Baranyi and Roberts was fitted at curve of the growth, previously obtained in Biochemical Engineering Laboratory, of W. viridescens in culture medium in six different isothermal temperatures (4 to 30 ° C). Were evaluated four secondary models to describe the influence of temperature on the growth parameters, which was best described by the model of the square root for µmáx parameter. The secondary model for the Ymax parameter was considered as the mean values obtained experimentally, independent of temperature, the temperature range studied. Based on the selected models, Baranyi and Roberts (1994) model was used to predict the growth of W. viridescens under non-isothermal conditions. The proposed model was validated with experimental data of W. viridescens growth in MRS medium under different temperature profiles involving temperatures above refrigeration temperature and temperature fluctuations in domestic refrigerators. The results showed that this predictive model has the possibility of being used to predict the shelf-life of meat products which deterioration occurs mainly as a result of W. viridescens growth.

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências Agrárias, Programa de Pós-Graduação em Aquicultura, Florianópolis, 2014.
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O presente trabalho objetivou avaliar os efeitos da suplementação da bactéria probiótica Weissella cibaria no trato intestinal e na saúde de surubins híbridos (Pseudoplatystoma corruscans e P. reticulatum ). Os 96 peixes foram distribuídos em 12 tanques circulares de 100 L, em sistema de recirculação de água. Os peixes foram alimentados com 3% da biomassa total durante 45 dias, sendo os peixes do tratamento alimentados com ração comercial suplementada com probiótico, e os peixes do grupo controle com ração comercial sem suplementação. O número de eritrócitos totais, trombócitos e linfócitos se apresentaram maiores nos peixes alimentados com a suplementação probiótica (p<0,05). A porcentagem de fagocitose, o título aglutinante e a concentração total de imunoglobulinas foram maiores nos peixes alimentados com a suplementação probiótica (p<0,05). Através de técnicas de microscopia de luz e eletrônica, foi possível verificar a alteração na microbiota autóctone dos peixes, além do incremento, daqueles que receberam suplementação probiótica, no comprimento e largura das vilosidades intestinais, do número de células caliceformes por vilo, além do perímetro dessas vilosidades. A bactéria W. cibaria foi capaz de colonizar e alterar a microbiota intestinal, assim como sua ultraestrura, além de modular os parâmetros hemato-imunológicos.

Giardiasis is a disease caused by the protozoan Giardia lamblia, non-invasive pathogenic parasite that infects 280 million people annually worldwide. The introduction of probiotics in the treatment of various diseases have increased in recent decades due to the benefits generated in the health of the host. Much of the probiotic strains with antimicrobial properties and immunomodulatory proven, are lactic acid bacteria that in microaerophilic. Given the above, we decided to evaluate the effect of probiotics on Giardia lamblia in experimental infections. Probiotics Bifidobacterium longum 51A were studied and Weissella paramesenteroides WpK4, this very last newly isolated and still in evidence phase of its probiotic function. We used 60 gerbils, males and females, divided into 6 groups: B. longum 51A with Giardia (BLi); group B. longum 51A without Giardia (BLC); W. paramesenteroides group with Giardia (WPi); W. paramesenteroides group without Giardia (WPC); infection control group (C); control group (CC). Animals received probiotic (B. longum 51A or W. paramesenteroides WpK4), 0.9% saline in the amount of 0.1 mL daily, and Giardia lamblia suspension by gavage. After 10 days of daily treatment with probiotics groups of animals BLi, WPi and Ci were infected with G. lamblia were euthanized on the seventeenth day after initiation of the experiment. For histological analysis, the duodenum of the small intestine was collected, fixed and designed to obtain sections stained with H & E and PAS. The villus height, crypt, the relationship crypt / villus, mucus area and area of immunostained trophozoites were calculated using the software installed in KS300 by Carl Zeiss image analyzer. The results were generated with GraphPad PRISM ® 4 software (GraphPad Software Inc.). In all tests were considered statistical difference values with significance level of 5% (p <0.05). The height of the intestinal crypts increased significantly in infected groups (Ci), (BLi) and (WPi) compared to the other experimental groups. We observed that the relationship crypt / villus was significantly higher in the infected group (Ci), (BLi) and (WPi) when compared to the others. The analysis of trophozoites immunomarked area showed a significant reduction in the number of trophozoites BLi and WPi groups in relation to Ci group. As for PAS positive area observed that in the group BLi the positive PAS regions in the crypts and villi was significantly higher compared to BLC groups, WPC, WPi. This study demonstrated that administration of probiotic bacteria (B. longum 51A and W. paramesenteroides) reduced the animal load infected with G. lamblia.
A giardíase é uma doença causada pelo protozoário Giardia lamblia, um parasito patogênico não invasivo que infecta anualmente 280 milhões de indivíduos em todo o mundo. A introdução de probióticos na terapêutica de diversas doenças têm aumentado nas ultimas décadas em função dos benefícios gerados na saúde do hospedeiro. Grande parte das linhagens probióticas com propriedades antimicrobianas e imunomoduladoras comprovadas, são bactérias ácido-láticas que em microaerofilia. Diante do exposto, decidimos avaliar a ação de probióticos sobre Giardia lamblia em infecções experimentais. Os probióticos estudados foram Bifidobacterium longum 51A e Weissella paramesenteroides WpK4, esta útima recém-isolada e ainda em fase de comprovação de sua função probiótica. Utilizaram-se 60 gerbils, machos e fêmeas, distribuídos em 6 grupos: B. longum 51A com Giardia (BLi); grupo B. longum 51A sem Giardia (BLc); grupo W. paramesenteroides com Giardia (WPi); grupo W. paramesenteroides sem Giardia (WPc); grupo controle infecção (Ci); grupo controle (Cc). Os animais receberam os probióticos (B. longum 51A ou W. paramesenteroides WpK4), salina a 0,9%, na quantidade de 0,1mL diariamente, e suspensão de Giardia lamblia via gavagem,. Após 10 dias de tratamento diário com os probióticos os animais dos grupos BLi, WPi e Ci, foram infectados com G. lamblia e eutanasiados no décimo sétimo dia após o início do experimento. Para análise histológica, a porção duodenal do intestino delgado foi coletada, fixada e destinada à obtenção de cortes corados com H & E e PAS. A altura das vilosidades, das criptas, a relação criptas/vilosidades, área de muco e área de trofozoítos imunomarcados, foram calculadas através do software KS300 instalado no analisador de imagens Carl Zeiss. Os resultados foram produzidos com o software GraphPad Prism® 4 (GraphPad Software Inc.). Em todos os testes foram considerados como diferenças estatísticas os valores com nível de significância menor que 5% (p<0,05). A altura das criptas intestinais apresentou aumento significativo nos grupos infectados (Ci), (BLi) e (WPi) em relação aos outros grupos experimentais. Observamos que a relação cripta/vilosidade foi significantemente maior nos grupos infectados (Ci), (BLi) e (WPi) quando comparados aos demais. A análise da área de trofozoítos imunomarcados demonstrou redução significativa no número de trofozoítos dos grupos BLi e WPi em relação ao grupo Ci. Quanto a área de PAS positivo observamos que no grupo BLi as regiões PAS positiva nas criptas e vilosidades foi significantemente maior em relação aos grupos BLc, WPc, WPi. Este trabalho demonstrou que a administração de bactérias probióticas (B. longum 51A e W. paramesenteroides) reduziu a carga parasitária dos animais infectados com G. lamblia.

Les bactéries lactiques sont utilisées pour la fermentation d'aliments dans le but d'augmenter leur durée de conservation et d'améliorer leurs propriétés organoleptiques et nutritionnelles. Dans le but de diversifier l'offre d'aliments fonctionnels et de limiter les pertes en produit frais, nous proposons des produits fermentés à base de fruits ou légumes, riches en antioxydants et plaisants pour le consommateur. Pour cela, mes travaux de thèse ont été menés en deux étapes : caractériser la flore bactérienne lactique présente à la surface de fruits et de légumes cultivés à La Réunion, puis sélectionner des bactéries autochtones possédant certaines propriétés fonctionnelles. Ainsi, 77 bactéries lactiques isolées de papayes, de tomates et d'achards de la Réunion, appartenant aux genres Leuconostoc, Lactococcus, Weissella, Lactobacillus et Fructobacillus ont été caractérisées génétiquement et phénotypiquement. Parmi la grande diversité observée, certains isolats ont présenté des caractéristiques technologiques (conditions et vitesse de croissance, résistance aux stress environnementaux) et fonctionnelles (production d'exo-polysaccharides) intéressantes pour l'élaboration d'aliments fermentés, en particulier les isolats Weissellla cibaria 64 et 30, Leuconostoc pseudomesenteroides 60 et Lactobacillus plantarum 75. A partir de ces données, des étapes complémentaires de criblage ont été réalisées sur différents substrats (mangue, papaye, ananas, thé vert, thé noir) fermentés, en examinant leurs propriétés organoleptiques et leurs activités antioxydantes. Deux isolats, Lc. pseudomesenteroides 12b et W. cibaria 64, ont augmenté significativement la teneur en composés phénoliques et l'activité antioxydante de jus d'ananas Victoria au cours d'une fermentation de 48h. Le produit fermenté se conserve 16 jours au froid sans aucune altération et conserve l'ensemble de ses bénéfices nutritionnels. Une odeur et des goûts typiques ont été détectés dans les boissons fermentées obtenues. L'augmentation de l'activité antioxydante observée au cours de la fermentation résulte probablement du métabolisme des composés phénoliques par les bactéries. L'identification des molécules produites et des enzymes impliquées est nécessaire afin de comprendre les mécanismes mis en jeu
Lactic acid bacteria are used for the production of large variety of fermented foods in order to enhance shelf-life together with improved organoleptic and nutritional properties. In order to create new functional foods and to reduce fresh product waste, the objectives of my PhD were to develop and characterize fruit or vegetables fermented foods, rich in antioxidants and pleasant for consumers. To reach these goals, my PhD work was split in two steps: 1- characterization of lactic bacteria present on fruits and vegetables grown at Reunion Island and 2- selection of autochthonous bacteria with functional properties. Thus, 77 lactic bacteria from genera Leuconostoc, Lactococcus, Weissella, Lactobacillus and Fructobacillus were isolated from papaya, tomato and sliced cabbage from Reunion Island. They were genetically and phenotypically characterized. A huge diversity in term of genetic and phenotypic characteristics was determined. Furthermore, several isolates exhibiting specific technological and functional properties (growth rate, resistance to environmental stress, production of exopolysaccharides) were identified. These isolates, potentially useful for the production of fermented foods, were Weissella cibaria 64 and 30, Leuconostoc pseudomesenteroides 60 and Lactobacillus plantarum 75. Then, further screening steps were performed on different food substrates (pineapple, mango, papaya, tea infusions) in order to select isolates able to improve antioxidant and organoleptic properties. Two isolates, W. cibaria 64 and Lc. pseudomesenteroides 12b were shown to significantly enhance the phenolic content and the antioxidant activity of Victoria pineapple juice after a 48h-fermentation. The fermented products exhibited a good shelf-life of 16 days without alteration and preserved nutritional benefits. Characteristic odour and tastes were detected in the fermented drinks. The increase in antioxidant activity observed during fermentation was probably the consequence of a depolymerization of phenolic compounds. Further works are required to identify the composition changes over fermentation and to reach a better understanding of the mechanisms involved

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A indústria alcooleira no Brasil perde anualmente vários milhares de reais devido a episódios de contaminação produzidos por bactérias. Além disso, outros milhares são gastos com o uso de antibióticos industriais para a contenção dessas contaminações. Infelizmente, não há ainda um catálogo completo de informações sobre todas as possíveis espécies bacterianas envolvidas na contaminação industrial, seus efeitos no processo e os fatores que levam a essas contaminações. Com a crescente demanda por fontes de energia renováveis, principalmente o etanol, grandes esforços têm sido feitos para aumentar a sua produção. Este trabalho tem como objetivo principal identificar as bactérias lácticas que entram no processo de fermentação alcoólica através do caldo de cana-de-açúcar com a utilização de ferramentas moleculares para uma rápida e eficiente identificação. Para foram realizadas coletas em quatro destilarias do Nordeste do Brasil durante a safra de 2007/2008. A identificação dos isolados foi realizada através da técnica ARDRA e pela análise do sequenciamento de DNA dos genes pheS e 16S. Os resultados obtidos mostram que os lactobacilos são os principais contaminantes do processo, com o Lactobacillus fermentum se destacando entre as demais. Também foi observado que entram no processo bactérias dos gene Weissella e Leuconostoc, com destaque para a W. confusa e o Leuc. mesenteroides que foram identificadas em três das destilarias estudadas. Além da identificação, os isolados de W. confusa e o Leuc. Mesenteroides foram tipados pelo REP-PCR com o primer (GTG)5. Os resultados mostraram que, além da diversidade de espécies bacterianas detectadas, existe uma diversidade de linhagens dessas duas principais espécies no processo. A espécie Leuc. mesenteroides tem sido apontada como contaminante em processo de fermentação alcoólica industrial, sendo algumas linhagens capazes de causar a formação de dextrana, podendo causar danos ao processo fermentativo. Desta forma se faz necessário trabalhos futuros com o fim de correlacionar os perfis destas espécies com possíveis danos à fermentação, para que com este conhecimento possa ser criadas novas estratégias de controle destes microorganismos.

Our large intestine is like a large metabolic organ colonised by microorganisms. Beneficial probiotic bacteria are of interest since they might metabolise certain prebiotic carbohydrates and produce metabolites that are suggested to promote health and prevent diseases.   Strains of Weissella have proven probiotic properties since they, for example, show ability to metabolise prebiotic oligosaccharides, are resistant to a low pH (pH 2-3) and bile salt. In a previous project, six new strains of Weissella were isolated from Indian fermented food and vegetables, and four of them, including strain 92, were able to ferment xylooligosaccharides and form short chain fatty acids (SCFA), especially acetic acid. This strengthened the probiotic potential of these strains.   The aim of this project was to see if previously untested oligosaccharides (arabinooligosaccharides (AOS), laminarioligosaccharides (LOS) and chitooligosaccharides (COS)) could be metabolised by Weissella strain 92.   This study includes the following steps; cell growth in MRS (De Man, Rogosa and Sharpe) medium on different carbohydrates measured with spectrophotometer, pH measurement (analysing the difference of MRS medium (pH 6.42) pre and post cell growth, where reduced pH indicates acid production), and analysis of fermentation products (including SCFA (short chain fatty acids, e.g. acetic acid, butyric acid, propionic acid), lactic acid and ethanol) with an HPLC (high-performance liquid chromatography) instrument.   This research study has shown that Weissella strain 92 produces acetic and butyric acid as a consequence of use of AOS, LOS and COS, this indicates that the oligosaccharides are prebiotic and emphasizes the probiotic potential of Weissella strain 92.

Trabajo donde se describe la ocurrencia de una enfermedad emergente en poblaciones de trucha arcoíris de México. Se detallan los signos clínicos y lesiones macroscópicas observadas en peces afectados. Se describe la importancia epidemiológica de esta enfermedad, hasta ahora solo reportada en China, Brasil y en los Estados Unidos de Norteamérica.
Dentro del grupo de las denominadas bacterias acido lácticas (BAL) se encuentra la bacteria Weissella ceti, una bacteria emergente identificada como patógena que afecta a la trucha arcoíris Oncorhynchus mykiss presentándose en forma septicémica. En México, se han presentado mortalidades en granjas productoras de trucha arcoíris con signos sugerentes a la enfermedad denominada Weissellosis. En el presente estudio se aislaron bacterias cocobacilares de organismos con signología aparente de enfermedad que incluyen: exoftalmia, opacidad corneal, lesiones hemorrágicas en ojos, boca, aletas pectorales, hemorragias multifocales en hígado y cerebro. Los aislados se caracterizaron bioquímicamente como bacterias Gram positivas, oxidasa y catalasa negativas, hemolíticas, no móviles, con actividad fermentadora a partir de los carbohidratos dextrosa, glucosa, maltosa y trehalosa. La bacteria Weissella ceti aislada de los organismos afectados fue genéticamente caracterizada por medio del uso de PCR convencional utilizando primers específicos de un sitio de adhesión al colágeno identificado únicamente en esta especie. Las lesiones histológicas que se evidenciaron en las truchas arcoíris afectadas por esta bacteria fueron en ojo separación de fibras del estroma y hemorragia en la coroides, hepatitis necrótica multifocal con hepatocitos en degeneración y meningitis y congestión en cerebro. Este es el primer reporte de la bacteria en cuadros septicémicos en truchas arcoíris en México.
4489/2018/CI. Caracterización bioquímica y molecular de Streptococcus spp obtenidas de trucha arcoíris (Oncorhynchus mykiss)

Weissella est un genre bactérien appartenant au groupe de bactéries lactiques. Les souches appartenant au genre Weissella sont autochtones et vivent dans des environnements laitiers et non laitiers. Elles ont la capacité de produire divers composés d'intérêt pour l'industrie alimentaire. Ce travail de thèse a débuté par deux revues de littérature qui ont permis de mettre en relief la biologie mais aussi le potentiel d'application des souches Weissella pour les industries alimentaires et pharmaceutiques. En effet, certaines souches de Weissella se sont avérées être bénéfiques dans ce domaine en raison de leur capacité à produire des oligosaccharides non digestibles. Ce travail de thèse s'est poursuivi par l'étude de la biodiversité des souches isolées de fromages artisanaux de différentes régions du Brésil. Les analyses par les techniques PFGE et Rep-PCR ont permis de mettre des profils différents et donc des niches écologiques typiques. A la suite de cette analyse, nous avons sélectionné des candidats pour chaque groupe et procédé à leur caractérisation, comme leur potentiel antimicrobien contre des agents pathogènes d'origine(s) alimentaire(s). La plupart des candidats testés ont montré une activité inhibitrice contre ces germes pathogènes. Outre cette activité antagoniste, nous avons étudié le potentiel technologique de treize souches. Sur la base de tous ces résultats, nous avons retenu trois souches avec une bonne activité inhibitrice, la première est Weissella cibaria W25 (W25). Le génome de cette souche a été séquencé et assemblé. Ensuite, nous avons comparé le génome de notre souche W25 à celui de W. cibaria 110, connue pour produire la bactériocine weissellicine 110, et ceux de quatre autres souches de W. cibaria non productrices de bactériocines. Les analyses bio-informatiques ont montré que la souche W25 possède des gènes de bactériocines selon le logiciel AntiSmash ; laissant à penser la possibilité de produire deux bactériocines différentes. Nous avons par la même approche analysée les génomes des trois autres souches de Weissella à savoir W21, W25 et W42. Cette étude comparative a permis de localiser de l'ADN (phase de lecture ouverte) pouvant correspondre à une bactériocine putative identifiée comme de la classe_IIc. Les comparaisons de séquences nucléotidiques et en acides aminés de cette bactériocine putative avec celles disponibles dans la base de données NCBI n'ont pas permis de trouver des similitudes, laissant présager une nouvelle bactériocine non décrite à ce jour. Enfin, d'autres travaux expérimentaux ont été menées sur trois souches afin d''établir leur potentiel probiotique pour une application humaine et/ou animale. Il en ressort de cette étude que les trois souches de Weissella ne possèdent pas de gènes acquis de résistance aux antibiotiques, ni d'éléments génétiques mobiles dans leurs génomes. Ces souches se distinguent par une absence d'activité hémolytique et de facteur de virulence connu et sont dotées de système de défense CRISPR-Cas. En plus, elles ne présentent aucune capacité d'inflammation ou de cytotoxicité sur les cellules eucaryotes de type Caco-2. En particulier, la souche W21 présente d'excellents résultats d'adhésion aux cellules intestinales et se distingue par sa capacité d'exclusion d'un pathogène redoutable, en l'occurrence Staphylococcus aureus. Comme la majorité des probiotiques, aucune des trois souches n'a révélé des capacités de survie dans les conditions mimant le tractus gastro-intestinal, ce qui ne va pas limiter leur utilisation car des moyens de protection existent notamment l'encapsulation. En conclusion, ce travail a permis d'établir une première cartographie de la diversité et de la distribution des espèces de Weissella dans différents fromages artisanaux du Brésil. Cette étude a permis de montrer le potentiel de ces souches du genre Weissella à produire des bactériocines mais aussi à être utilisées comme probiotiques, voire comme cultures protectrices
.Weissella is a bacterial genus belonging to the lactic acid bacteria group. Strains belonging to the genus Weissella are indigenous, which are found in dairy and non-dairy environments. They have the ability to produce various compounds of interest for the food industry. This thesis work started with two literature reviews that highlighted the biology but also the potential application of Weissella strains for the food and pharmaceutical industries. Indeed, some Weissella strains have proven to be beneficial in this field because of their ability to produce non-digestible oligosaccharides. This thesis work continued then with the study of the biodiversity of strains isolated from artisanal cheeses from different regions of Brazil. Analyses by PFGE and Rep-PCR techniques have allowed us to identify different profiles and thus typical ecological niches. Following this analysis, we selected candidates for each group and proceeded to their characterization, such as their antimicrobial potential against foodborne pathogens. Most of the tested candidates showed an inhibitory activity against these pathogens. In addition to this antagonistic activity, we studied the technological potential of thirteen strains. Based on all these results, we selected three strains with good inhibitory activity, the first one is Weissella cibaria W25 (W25). The genome of this strain was sequenced and assembled. Then, we compared the genome of our strain W25 with that of W. cibaria 110, known to produce the bacteriocin weissellicin 110, and those of four other non-bacteriocin-producing strains of W. cibaria. Bioinformatics analyses showed that strain W25 possesses bacteriocin genes according to AntiSmash software; suggesting the possibility of producing two different bacteriocins. By the same approach, we analyzed the genomes of the three other strains of Weissella, namely W21, W25 and W42. This comparative study allowed us to locate DNA (open reading phase) that could correspond to a putative bacteriocin identified as class_IIc. Nucleotide and amino acid sequence comparisons of this putative bacteriocin with those available in the NCBI database did not reveal any similarities, suggesting a new bacteriocin not yet described. Finally, other experimental work was conducted on three strains in order to establish their probiotic potential for human and/or animal application. The results of this study show that the three Weissella strains do not possess acquired antibiotic resistance genes, nor mobile genetic elements in their genomes. These strains are characterized by the absence of hemolytic activity and known virulence factors and have no CRISPR-Cas defense system. In addition, they do not show any inflammatory or cytotoxic capacity on Caco-2 eukaryotic cells. In particular, the W21 strain shows excellent adhesion to intestinal cells and stands out for its ability to exclude a formidable pathogen, in this case Staphylococcus aureus. Like the majority of probiotics, none of the three strains revealed survival capacities in conditions mimicking the gastrointestinal tract, which will not limit their use because means of protection exist, notably encapsulation. In conclusion, this work allowed to establish a first snapshot of the diversity and distribution of Weissella species in different artisanal cheeses from Brazil. This study allowed to show the potential of these strains of the Weissella genus to produce bacteriocins but also to be used as probiotics or even as protective cultures

Tese (doutorado) - Universidade Federal de Santa Catarina, Centro Tecnológico, Programa de Pós-Graduação em Engenharia de Alimentos, Florianópolis, 2016.
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A bactéria ácido-láctica Weissella viridescens tem sido indicada como um dos principais micro-organismos deteriorantes de produtos cárneos. O crescimento de micro-organismos sob condições isotérmicas ou não-isotérmicas pode ser descrito por modelos matemáticos, cujos parâmetros são estimados com base em dados experimentais de crescimento, com valores que dependem da abordagem utilizada na estimativa. Na Modelagem em Duas Etapas (MDE), o ajuste dos modelos primário e secundário é sequencial e tem como base experimentos em condições isotérmicas, sendo esta a abordagem mais reportada na literatura. No entanto, os parâmetros dos modelos podem ser estimados simultaneamente com a Modelagem em Uma Etapa (MUE), com experimentos em condições isotérmicas ou não-isotérmicas. Para reduzir o tempo experimental, a quantidade e o custo de dados experimentais, e a incerteza dos parâmetros (intervalos de confiança), os experimentos em condições não-isotérmicas podem ser otimizados com o Delineamento Experimental Ótimo (DEO). Para isso, matrizes que quantificam a sensibilidade das respostas dos modelos em relação às variações nos valores dos parâmetros são otimizadas. O objetivo desta tese de doutorado foi aplicar o DEO para estimar os parâmetros do crescimento de W. viridescens em meio de cultivo e em presunto, com menores tempos, quantidade de dados experimentais e redução da incerteza dos parâmetros, quando comparado às abordagens MDE e MUE. Os experimentos para a estimativa dos parâmetros foram realizados em meio de cultivo em seis condições isotérmicas (para a MDE e MUE em condições isotérmicas, MUEiso); em quatro condições não-isotérmicas não-ótimas (para a MUE em condições não-isotérmicas, MUEnao); e em quatro condições não-isotérmicas delineadas de forma ótima (para o DEO). Um experimento adicional delineado de forma ótima foi utilizado para a validação dos parâmetros. Os experimentos nas cinco condições não-isotérmicas ótimas foram realizados também em presunto (DEOpres) para a estimativa dos parâmetros e validação. O modelo primário de Baranyi e Roberts e o modelo secundário da Raiz Quadrada foram utilizados para descrever o crescimento microbiano. Índices estatísticos foram utilizados para avaliar a qualidade dos ajustes dos modelos aos dados e a capacidade preditiva dos modelos. Em meio de cultivo, os parâmetros estimados com a MDE, a MUEiso e o DEO foram próximos uns dos outros, sendo que as incertezas dos parâmetros foram menores com a MUEiso e similares com a MDE e DEO. Entretanto, houve umagrande redução na quantidade de dados e tempo experimental necessários para estimar os parâmetros, de 196 dados e 3.540 horas na MDE e MUEiso para 60 dados e 588 horas no DEO. Além disso, os modelos resultantes do DEO apresentaram as melhores predições. Os parâmetros estimados na MUEnao foram distintos dos demais, com maiores incertezas, e os ajustes dos modelos resultaram em índices estatísticos insatisfatórios. O DEO foi utilizado também com sucesso na estimativa dos parâmetros de crescimento de W. viridescens em presunto. Os parâmetros estimados em presunto com DEO apresentaram incerteza similar aos parâmetros estimados em meio de cultivo, e os índices estatísticos confirmaram o bom ajuste dos modelos aos dados experimentais. Os modelos com os parâmetros estimados com o DEOpres apresentaram predições satisfatórias do crescimento microbiano também em presunto. Portanto, os resultados desta tese apontam que o DEO pode ser utilizado com sucesso para estimar os parâmetros do crescimento microbiano em meio de cultivo e em presunto, resultando em modelos com boa capacidade preditiva. Além disso, os parâmetros podem ser estimados com menores tempos e quantidade de dados experimentais, e menores incertezas dos parâmetros quando comparado às abordagens MDE e MUE.

Abstract : The acid-lactic bacteria Weissella viridescens has been indicated as one of the main spoilage microorganisms of meat products. The microbial growth under isothermal or non-isothermal conditions can be described by mathematical models, which parameters are estimated based on growth experimental data, with values that depend on the approach used in the estimation. In the Modeling in Two Step (MTS), the fitting of primary and secondary models is sequential, and has as base experiment under isothermal conditions, being the approach most reported in the literature. However, the parameters of the models can be estimated simultaneously with the Modeling in One Step (MOS) with experiments in isothermal or non-isothermal conditions. To reduce the experimental time, quantity and cost of experimental data, and the uncertainty of the parameters (confidence intervals), the experiments in non-isothermal conditions can be optimized with Optimal Experimental Design (OED). To do that, matrices which quantify the sensitivity of the model responses to the variations in the values of the parameters are optimized. The objective of this doctoral thesis was applying the OED to estimate the growth parameters of W. viridescens in culture medium and ham with lower time, quantity of experimental data and reduction of parameter uncertainty when compared to MTS and MOS approaches. The experiments for the parameter estimation were performed in culture medium in six isothermal conditions (for MTS and MOS in isothermal conditions, MOSiso), in four non-optimal non-isothermal conditions (for MOS in non-isothermal conditions, MOSnon), and in four optimal non-isothermal conditions (for OED). One additional experiment optimally designed was used for the validation of the parameters. The experiments in the five optimal non-isothermal conditions were also performed in ham (OEDham) for the estimation of the parameters and validation. The Baranyi and Roberts primary model and the Square Root secondary model were used to describe the microbial growth. Statistical indexes were used to assess the quality of the model fitting to the data and the predictive ability of the models. In culture medium, the parameters estimated with the MTS, the MOSiso and the OED were near each other, in which the uncertainty of the parameters were lower with the MOS and similar with the MTS and OED. However, there were a great reduction in the quantity of data and experimental time needed to estimate the parameters, from 196 data and 3,540 hours in MTS and MOSiso to 60 data and 588 hours in OED. Furthermore, the models resulting of OED presented the greatestpredictions. The parameters estimated in MOSnon were distinct from others, with greater uncertainty, and the model fitting resulted in unsatisfying statistical indexes. The OED was also used successfully in the estimation of the growth parameters of W. viridescens in ham. The parameters estimated in ham with OED presented similar uncertainty to the parameters estimated in culture medium, and the statistical indexes confirmed the good fitting of the models to the experimental data. The models with the parameters estimated with the OEDham presented satisfactory predictions of the microbial growth also in ham. Therefore, the results of this thesis point out that the OED can be successfully used to estimate the microbial growth parameters in culture medium and in ham, resulting in models with good predictive ability. Furthermore, the parameters can be estimated with lower time and quantity of experimental data, and lower uncertainty of parameters when compared to the MTS and MOS approaches.

Streptococcus dysgalactiae and Weissella ceti have been considered emergent pathogens for Brazilian Nile tilapia (Oreochromis niloticus) and rainbow trout (Onchorhynchus mykiss) farming, respectively. Both diseases cause great economic losses in fish farms. Disease prevention by vaccination is a fundamental method for pathogen control in aquaculture. The aims of this study were to evaluate the genetic diversity of S. dysgalactiae and W. ceti strains isolated from distinct geographic origins and to develop efficient vaccines against these pathogens. Different methods (REP-PCR, ERIC-PCR and PFGE) were used for the genetically characterization of strains of both bacteria, and sodA gene sequencing was also tested for genotyping of S. dysgalactiae isolates. Aqueous-based whole cell killed bacterin and oil-adjuvanted vaccine were tested. The PFGE was the best genotyping method for S. dysgalactiae and detected three different genetic profiles among the evaluated strains. The genetic variability of patterns was clearly associated with the geographic origin of isolates. According to the methods used, Brazilian W. ceti isolates was found to be highly homogeneous with a clonal population. Vaccines formulated with whole cell bacterin emulsified in oil adjuvant provided significant protection against S. dysgalactiae (RPS = 92,5) and W. ceti (RPS = 92,0) infections in Nile tilapia and rainbow trout, respectively. The present results provide scientific information for development of adequate methods to control two emerging infectious diseases that threaten Brazilian fish farming industry.
As bactérias Streptococcus dysgalactiae e Weissella ceti são consideradas patógenos emergentes para a piscicultura nacional, causando perdas significativas nas cadeias produtivas de tilápia do Nilo (Oreochromis niloticus) e truta arco-íris (Onchorhynchus mykiss), respectivamente. Os objetivos do presente estudo foram avaliar a diversidade genética de amostras de S. dysgalactiae e W. ceti provenientes de regiões geográficas diversas e desenvolver vacinas eficazes na proteção contra esses patógenos. Diferentes métodos (REP-PCR, ERIC-PCR e PFGE) foram utilizados para caracterização genética das bactérias. A análise do sequenciamento do gene sodA foi utilizada somente para a genotipagem de amostras de S. dysgalactiae. Duas diferentes vacinas foram testadas: bacterina e bacterina emulsificada com adjuvante Montanide. A técnica de PFGE foi a mais eficaz para a genotipagem dos isolados de S. dysgalactiae, detectando três perfis genéticos diferentes. Os padrões genéticos estão associados com a origem geográfica dos isolados. De acordo com os métodos testados, as amostras brasileiras de W. ceti apresentam alta similaridade genética, sendo consideradas clonalmente relacionadas. As vacinas formuladas a partir de células inteiras inativadas (bacterina) emulsificadas em adjuvante oleoso conferiram proteção frente as infecções por S. dysgalactiae (RPS = 92,5) e W. ceti (RPS = 92,0) em tilápia do Nilo e truta arco-íris, respectivamente. Os resultados do presente projeto fornecem informações científicas e ferramentas fundamentais para o controle de duas doenças infecciosas emergentes da piscicultura nacional.

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro Tecnológico, Programa de Pós-Graduação em Engenharia de Alimentos, Florianópolis, 2017.
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As bactérias ácido lácticas (BAL) são consideradas os principais microrganismos deteriorantes de produtos cárneos cozidos, embalados à vácuo, mantidos sob refrigeração. A principal flora responsável pela deterioração na carne fresca e produtos lácteos, durante a armazenagem refrigerada aeróbia, é composta por espécies de Pseudomonas. Essas bactérias podem causar alterações como sabores estranhos, descoloração, diminuição do pH e produção de limo. Devido à demanda dos consumidores por embalagens de alimentos mais naturais, potencialmente biodegradáveis e recicláveis, os estudos têm focado na incorporação de compostos antimicrobianos naturais, como os óleos essenciais (OEs) nos materiais de embalagens biodegrádaveis. O óleo essencial de orégano (OEO) tem sido utilizado como agente antimicrobiano natural visando aumentar a vida útil de produtos alimentares. O acetato de celulose vem ganhando destaque para a utilização em embalagens, por ser um polímero biodegrádavel e por suas diferentes aplicabilidades. Frente ao exposto, o objetivo geral deste trabalho foi avaliar a ação antimicrobiana de filmes ativos de acetato de celulose incorporados com diferentes concentrações de OEO no crescimento de Weissella viridescens e Pseudomonas fluorescens, para posterior aplicação em presunto nas concentrações mais efetivas. Para a avaliação do efeito antimicrobiano do OEO foram determinadas a Concentração Mínima Inibitória (CMI) (fase líquida), Dose mínima inibitória (DMI) (fase vapor) e efeito bacteriostático e bacterida sobre o crescimento de W. viridescens e P. fluorescens. O efeito dos filmes ativos, incorporado com diferentes concentrações de OEO, sobre o crescimento de W. viridescens e P. fluorecens foi avaliado em fase vapor. O presunto foi caracterizado quanto ao seu pH, atividade de água e concentração de cloreto de sódio, que encontravam-se dentro da faixa ótima de desenvolvimento de BAL descrita pela literatura. Foi avaliado o crescimento de W. viridescens e P. fluorescens no presunto a 8 °C, com e sem adição de filme ativo nas concentrações de 10 e 15%. O filme incorporado com 15% de OEO levou a um aumento da vida útil em 7 dias. A determinação das propriedades mecânicas dos filmes (Tensão de ruptura, Elongação e Módulo de Young) foi realizada nas concentrações mais efetivas (10 e 15% de OEO) sobre as bactérias estudadas, obtendo-se uma diferença significativa em relação ao controle (sem óleo) apenas pra a elongação. Os resultados mostraram que os filmes ativos incorporados com OEO se mostraram eficazes no controle do crecscimento das bactérias estudadas, mesmo quando aplicados no presunto, podendo ser uma boa alternativa para prolongar a vida útil do alimento.
Abstract : Lactic acid bacteria are considered the main spoilage microorganisms in cooked meat products, vacuum packed, keep under refrigeration. The main flora responsible for deterioration in fresh meat and dairy products, during aerobic refrigeration, is composed of Pseudomonas species. These bacteria can result in changes such as foreign flavors, discoloration, decreased pH and limo production. Due to consumer demand for more natural, potentially biodegradable and recyclable food packaging, studies have focused on the incorporation of natural antimicrobial compounds, like essential oils on biodegradable packaging materials. Oregano essential oil (OEO) has been used as a natural antimicrobial agent to increase the shelf life of food products. Cellulose acetate has been gaining attention for its use in packaging, because it is biodegradable and due its different applications. Thus, the aim of this study was evaluated the antimicrobial activity of cellulose acetate films incorporated with different concentrations of the OEO on Weisella viridescens and Pseudomonas fluorescens growth, for latter application in ham (most effective concentrations). To evaluated the antimicrobial effect of OEO, it was determined the minimal inhibitory concentration (CMI) (liquid phase), minimal inhibitory dose (DMI) (vapor phase) and bacteriostatic and bactericidal effects on W. viridescens and P. fluorescens growth. The effect of active films, incorporated with different concentrations of the OEO, on W. viridescens and P. fluorescens growth was evaluated on vapor phase. The ham was characterized in terms of pH, water activity and sodium chloride concentration, and these were within the optimal range of BAL development described in the literature. The growth of W. viridescens and P. fluorescens was evaluated in ham at 8 °C, with and without active film addition, at concentrations of 10 and 15%. The film incorporated with 15% of OEO led to an increase of the shelf life of 7 days. The determinations of the mechanical properties of the films were performed at the most effective concentrations (10 and 15% OEO) on the studied bacteria, obtaining the rupture stress (TR), elongation (E) and Young's Modulus (MY). A significant change was observed from control only for elongation. The obtained results showed that the films incorporated with the OEO were effective on the studied bacteria growth, even when applied on ham, and could be a good alternative to prolong the shelf life of the food.

Deoxynivalenol (DON) contamination of grain destined for animal feeds is a major toxicological risk to monogastrics and is suspected of restricting productivity in ruminants. Whereas bacterial additives have been developed that can detoxify DON in the rumen and lower intestine, there are currently no commercial inoculants able to perform this task in crimped grain (CG) silage, a regionally important method of moist grain preservation based on homo- and heterofermentative lactic acid bacteria or chemical additives. Determining whether this ensiling process alongside the action of detoxifying bacteria has the potential to remove DON in CG prior to ingestion, was explored in mini-silo ensiling experiments. CG was heat treated (100 °C, 60 min) or ensiled fresh in triplicate 50 g silos, spiked with 5 mg/kg DON and inoculated with lactic acid bacteria derived from wild birds, natural epiphytic inoculants and commercially sourced silage additives (21 d). DON recovery was only significantly reduced (31.2 ± 14.4% recovery, p < 0.001, n= 30) by heat treatment, as determined by IAC-RP-HPLC-UV. Bacterial assemblage analysis by 16S rRNA PCR-DGGE-SEQ identified Weissella cibaria, Pantoea agglomerans, Bacillus subtilis, B. licheniformis and Hafnia alvei as candidate detoxification agents, of which W. cibaria and H. alvei decreased DON recovery in vitro (11.3 and 6.2% recovery respectively, p < 0.05, n = 18), which translated to inoculated W. cibaria yielding a decrease in DON recovery (67.2± 14.4%, 28 d) in naturally contaminated crimped wheat (13.5 ± 1.0 mg/kg, 35-40% moisture, p < 0.05, n =15). As W. cibaria is a lactic acid bacteria already associated with fermented CG by default it has promise as a novel DON detoxification agent in CG silage. DON is however just one of many hepatotoxic co-contaminants. Retrorsine, a DNA-crosslinking pyrrolizidine alkaloid derived from Ragwort (Senecio sp.) was investigated for interactive toxicity with DON in an in vitro co-exposure experiment. HepG2 cells were exposed to Log10 multifactorial binary exposures for 48 h followed by a suite of assays to elucidate mechanisms of interactive cytotoxicity, genotoxicity and modulation of the proteome. Retrorsine was tentatively confirmed to form DNA/protein crosslinks in the comet, micronucleus and crosslinking assays, whilst DON was found to potently induce cytotoxicity and apoptosis. Co-exposure yielded a complex toxicity response, with low doses yielding antagonistic effects and high doses trending towards additive effects, although DON dose was generally the principle component. The difficulties associated with undertaking an interactive toxicity study where both toxins have multiple metabolic and cellular targets are highlighted.

Un modèle d'étude original a été établi afin d'étudier les phénomènes de résistance aux bactériocines de bactéries lactiques (petits peptides à activité antimicrobienne). Il comprend les deux mésentérocines produites par Leuconostoc mesenteroides subsp. Mesenteroides FR 52, quatre souches parentales (SP) issues des genres Leuconostoc ou Weissella, sensibles ou résistantes à ces peptides, et quatre résistants induits (RI) issus des trois souches parentales sensibles. L'étude du mode d'action de ces bactériocines a mis en évidence des phénomènes de synergie même vis-à-vis des souches uniquement sensibles à une seule bactériocine. Les deux mésentérocines induisent des modifications physiologiques chez les souches présentant une résistance naturelle ou induite, comme une augmentation de la teneur en acides gras rigidifiants et une plus grande résistance à certains antibiotiques. Aucune différence significative n'a été observée entre les SP et leur RI associé(s) dans les teneurs en acides gras membranaires. Les RI présentent des modifications pariétales dans leur morphologie cellulaire et leur sensibilité aux antibiotiques et au lyzozyme. Il n'a cependant pas été possible de conclure si ces différences étaient la cause ou les conséquences du phénotype de résistance.

碩士
國立高雄海洋科技大學
水產養殖研究所
101
In this study, various Weissella sp. strains isolated from fish gut were first tested their antimicrobial activity against the pathogen Streptococcus iniae by using the paper disk agar diffusion assay.The results showed that Weissella cibaria 981025mfic2, 981101spic2 and Weissella paramesenteroides 981213mfia1, 981213spi1 have higher antimicrobial activity. Therefore the four Weissella sp. strains were added into the feed to investigate their effects on the the growth, immune response and resistance against S. iniae in larvae and juvenile giant seaperch (Lates calcarifer) . After providing the feed containing 108 cfu g-1 of four Weissella sp. strains respectively for 14 days, we measured the growth rate of the giant seaperchlarvae and juveniles, evaluated the immune response of the giant seaperch juvenile, the survival rate of juvenile after intraperitoneal injection of S. iniae .The results showed that after 14 days feeding with Weissella sp. 981025mfic2, 981101spic2 containing feed, the growth rate of giant seaperch larvae feed was significant higher than the control growth (p <0.05). All the juveniles fed with Weissella sp. containing feed for 14 days had higher growth rate and the 981213spi1group especially had significant difference (p<0.05). The results of immune response evaluation demonstrated that the juvenile fed with Weissella sp. containing feed for 14 days had higher respiratory burst activity and lysozyme activity than those in the control group. For the respiratory burst activity assay, of the juvenile in 981213spi1 group had significant higher activity (p <0.05) and for the lysozyme activity assay, the juvenile in 981025mfic2 and 981213spi1 groups had significant differences (p <0.05). Seven days post intraperitoneal injection with S. iniae 103 CFU/fish, the experimental group 981025mfic2, 981101spic2, 981213mfia1 and 981213spi1 had higher survival rate, 50, 55, 55, 60% respectively, than the control group (40%). The results of bacterial count in the intestine of the experimental juvenile showed that after 14 days feeding with the Weissella sp. containing feed, the total number of bacteria in each experimental group were 4.61x105, 3.75x105, 7.30x106, 8.38x105 CFU/ml respectively and in the control group was7.79x105 CFU/ml. The lactic acid bacteria count using MRS agar in each experimental group were 4.12x104, 7.28x103, 5.73x104 CFU/ml and 4.64x104 CFU/ml respectively and no lactic acid bacteria was detected in the control group. However, the number of lactic acid bacteria in each experimental groups were gradually decreased after stopping Weissella sp. containing feed. Some giant seaperch juvenile didn’t contain lactic acid bacteria in the intestine after 4 or 5 days stopping Weissella sp. containing feed. In conculsion, feed containing W. cibaria (981025mfic2, 981101spic2) and W. paramesenteroides (981213mfia1, 981213spi1) can enhance the growth of Lates calcarifer larvae, and juveniles. Increase the respiratory burst and lysozyme activity in the juvenile and reducing the mortality after infected by S. iniae. However, these experimental bacteria cannot maintained in the intestine of giant seaperch for long time. It need to be supplemented with these intestinal lactic acid bacteria for maintaining a constant amount.

碩士
銘傳大學
生物科技學系碩士班
99
Weissellcin 110 is a newly discovered bacteriocin in Weissella cibaria 110. In the previously study, the partcial amino acid sequence of weissellcin 110 has been illustrated, however, the full-length sequence of this bacteriocin is still lacking. In current thesis, the attempt to discovery the DNA sequencing of both flanking regions neighboring the known sequence region, that included the 5’ and 3’ sequence unknown regions of wessellcin 110, and the genes neighbored. Two strategies that (1) traditional inversed PCR with restriction enzyme and T4 DNA ligase pretreated circular genomic fragmented template (2) newly designed PCR with random and ATCG sequence -based linker primer was introduced to deal with the problem. The current data demonstrated that both two strategies are work to amplify a constant PCR product. The following cloning and sequencing experiments are proceeding.

碩士
銘傳大學
生物科技學系碩士班
106
In this study, lactic acid bacteria (LAB) isolates were characterized and identified from litchi fruit. Strain Weissella confusa E isolated from litchi fruit was found to have bacteriocin-producing ability. The examination of characteristic of bacteriocin produced by W. confusa E was performed. During the initial phase of research, based upon 16S rDNA gene sequence, classification and identification of LAB was done through 16S rDNA RFLP (restriction fragment length polymorphism) and 16S rDNA sequencing. On the other hand, rpoA RFLP was used in order to differentiate the two high-affinity LAB: Leuconostoc mesenteroides and Leu. Pseudomesenteroides. Furthermore, purification of the bacteriocin that produced by W. confusa E was performed with the use of FPLC (Fast protein liquid chromatography), sep-pak C18 cartridges, and RP-HPLC (Reversed-phase High performance liquid chromatographic). Characteristic of the bacteriocin was further examined The 104 LAB isolated from litchi fruit were initially divided into five groups and Leuconostoc sp. was the most common species found. The results revealed that the restriction endonuclease AccII or AluI could cleave rpoA gene fragment, which could distinguish Leu. Mesenteroides and Leu. Pseudomesenteroide efficiently. Based on the bacteriologic test analysis, the molecular size of the bacteriocin, as determined by MALDI-TOF MS, revealed a distinctive peak corresponding to a molecular mass of 3426.7 Da. The data indicated the inhibitory activities of bacteriocin against Listeria monocytogenes. Because of the lack of identical results were found in the previous studies and its different molecular size compared to known bacteriocins from Weissella confusa, a novel bacteriocins were identified in this study.