Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.

Thesis (MSc Food Sc)--University of Stellenbosch, 2011. / ENGLISH ABSTRACT: The genus Cronobacter (Enterobacter sakazakii) contains opportunistic pathogens that can cause a severe form of neonatal meningitis, necrotising enterocolitis and septicaemia. Cronobacter infections have been reported in all age groups, however, immunocompromised infants are more susceptible to these infections. Furthermore, Cronobacter strains have been reported to show differences in sensitivity to antibiotics and virulence. These differences led to the reclassification of Cronobacter and currently the genus contains five distinct species, namely Cronobacter sakazakii, Cronobacter malonaticus, Cronobacter turicensis, Cronobacter dublinensis and Cronobacter muytjensii. As this reclassification was only accepted recently, there are not many typing methods optimised for differentiation between the five Cronobacter species. Typing of Cronobacter strains are important as the species may be diverse regarding their virulence. Cronobacter strains have been isolated from infant formula milk (IFM), the environment of an IFM processing facility and fresh produce in South Africa. However, little is known about the phylogeny and prevalence of these strains. The aim of this study was to classify 24 South African Cronobacter strains (previously identified as E. sakazakii) and to evaluate the phylogeny of the isolates based on the 16S ribosomal RNA (rRNA) and rpoA genes. All 24 South African strains were identified as Cr. sakazakii despite a wide variety of isolation sources. Other studies have also found that irrespective of the isolation source, the majority of Cronobacter strains are identified as Cr. sakazakii. The South African strains were found to be phylogenetically closely related. However, two distinct clusters separated at a 93 % confidence level were observed in the Cr. sakazakii group based on the 16S rRNA gene analysis. Strains of Cr. sakazakii, Cr. dublinensis, Cr. turicensis and Cr. muytjensii were differentiated from each other with sequence data of the 16S rRNA and rpoA genes, but it was not possible to differentiate between Cr. sakazakii and Cr. malonaticus. The phylogram based on the rpoA gene sequences did separate Cr. malonaticus and Cr. sakazakii strains, however, the clusters were separated with a low bootstrap value of 70 %. Phylogenetic analysis based on the rpoA and 16S rRNA genes were, therefore, not sufficient to distinguish between all the Cronobacter species. The sequence data of these two genes can be used to differentiate between the Cronobacter strains when used in combination with malonate utilisation analysis. A PCR-RFLP method was subsequently developed to facilitate the simultaneous differentiation between all five Cronobacter species. The PCR-RFLP assay was based on the amplification of the rpoB gene followed by the combined digestion with restriction endonucleases Csp6I and HinP1I. Unique profiles for each of the five Cronobacter species were obtained and it was also possible to differentiate between Enterobacteriaceae and Cronobacter strains. Furthermore, two strains which were identified as Cr. sakazakii with sequencing based on the 16S rRNA and rpoA genes had PCR-RFLP profiles identical to that of Cr. malonaticus. Sequencing based on the rpoB gene and additional biochemical analysis with malonate broth confirmed the identities of these two strains as Cr. malonaticus. This PCR-RFLP assay is, therefore, an accurate typing method that ensures rapid differentiation between the five species of Cronobacter. / AFRIKAANSE OPSOMMING: Die Cronobacter genus (Enterobacter sakazakii) bevat opportunistiese patogene wat 'n ernstige vorm van neonatale meningitis, enterokolitis en septisemie kan veroorsaak. Cronobacter infeksies is al in alle ouderdomsgroepe aangemeld, maar immuungekompromitteerde babas is die meeste vatbaar vir hierdie infeksies. Verder toon Cronobacter spesies verskille in virulensie en sensitiwiteit vir antibiotika. Hierdie verskille het gelei tot die herklassifikasie van Cronobacter en tans bestaan die genus uit vyf afsonderlike spesies, naamlik Cronobacter sakazakii, Cronobacter malonaticus, Cronobacter turicensis, Cronobacter dublinensis en Cronobacter muytjensii. Aangesien hierdie herklassifikasie slegs onlangs aanvaar is, is daar nie baie metodes wat geskik is vir onderskeiding tussen die vyf Cronobacter spesies nie. Onderskeiding tussen Cronobacter spesies is belangrik omdat die spesies verskillend kan wees met betrekking tot hulle virulensie. Cronobacter is geisoleer uit baba formule melk (BFM), die omgewing van 'n BFM fabriek en vars produkte in Suid-Afrika. Daar is egter nie baie bekend oor die filogenie en voorkoms van hierdie isolate nie. Die doel van hierdie studie was om 24 Suid-Afrikaanse Cronobacter stamme (voorheen geïdentifiseer as E. sakazakii) te klassifiseer en die filogenie van die isolate te evalueer gebaseer op die 16S ribosomale RNS (rRNS) en rpoA gene. Al 24 Suid-Afrikaanse stamme is geïdentifiseer as Cr. sakazakii ten spyte van 'n wye verskeidenheid isolasie bronne. Ander studies het ook gevind dat, ongeag die isolasie bron, die meerderheid van Cronobacter stamme as Cr. sakazakii geïdentifiseer word. In hierdie studie is gevind dat die Suid-Afrikaanse stamme filogeneties nou verwant is. Op grond van die 16S rRNA geen analise is die Cr. sakazakii stamme egter in twee afsonderlike groepe gedeel met 'n 93% vertrouens vlak. Dit was moontlik om stamme van Cr. sakazakii, Cr. dublinensis, Cr. turicensis en Cr. muytjensii van mekaar te onderskei met die DNS volgorde data van die 16S rRNA en rpoA gene, maar geen onderskeid tussen Cr. sakazakii en Cr. malonaticus stamme was moontlik nie. Die filogram gebaseer op die rpoA DNS volgorde data het aparte takke vir Cr. malonaticus en Cr. sakazakii stamme getoon, maar die twee takke is met ‘n lae vertrouens waarde van slegs 70 % geskei. Filogenetiese analise gebaseer op die rpoA en 16S rRNA gene is dus nie voldoende om te onderskei tussen al die Cronobacter spesies nie. Die DNS volgorde data van hierdie twee gene sou egter gebruik kon word om te onderskei tussen die Cronobacter spesies wanneer dit gebruik word in kombinasie met malonaatbenutting-analises. 'n Polimerase ketting reaksie (PKR) beperkings fragment lengte polimorfisme (BFLP) metode is ontwikkel om die gelyktydige onderskeiding tussen al vyf Cronobacter spesies te fasiliteer. Die PKR-BFLP metode is gebaseer op die vermeerdering van die rpoB geen gevolg deur die gesamentlike vertering met die beperkingsensieme, Csp6I en HinP1I. Unieke profiele vir elk van die vyf Cronobacter spesies is verkry en dit was ook moontlik om tussen Enterobacteriaceae en Cronobacter spesies te onderskei. Verder het twee stamme wat as Cr. sakazakii geïdentifiseer is met DNS volgordebepaling van die 16S rRNA en rpoA gene, PKR-BFLP profiele identies aan dié van Cr. malonaticus getoon. DNS volgordebepaling van die rpoB geen en ‘n addisionele biochemiese toets met malonaat sop het die identiteit van hierdie twee stamme as Cr. malonaticus bevestig. Hierdie PKR-BFLP is dus 'n akkurate metode wat vinnige onderskeid tussen die vyf spesies van Cronobacter kan verseker.

Thesis (MSc Food Sc)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: The consumption of game meat and its by-products is increasing locally and internationally. The increase in consumption requires research that is focused on the microbiological quality of game meat. The harvesting and slaughter process of springbok carcasses revealed the presence of bacterial contamination. Swab samples taken after skinning portrayed a presence of Escherichia coli (E. coli) and Enterobacteriaceae. Springbok carcasses swabbed after chilling indicated aerobic bacteria, Clostridium spp. and lactic acid bacteria. In contrast, swab samples taken at the evisceration’s incision area tend to be lower in counts when compared to swab samples taken after skinning and after chilling. Bacterial contamination was linked to poor hygienic practices during the harvesting and slaughter process. Results showed a need for the investigation of the slaughter process. To evaluate the slaughter process’s impact on the microbial quality of game carcasses, black wildebeest (Connochaetes gnou) carcasses were sampled throughout the slaughter process. Before skinning, aerobic bacteria, Enterobacteriaceae, and E. coli were enumerated from hide samples, counts ranged from 0.92 to 7.84 log cfu/g. after skinning, bacterial counts ranged from 0.93 to 6.12 log cfu/g and further decreased after chilling. Clostridium spp. counts increased after skinning, however, statistical analysis detected no significant differences between counts. Salmonella spp. was not detected. The results indicate that bacterial contamination does occur during the slaughter process. Hygienic status during the production of game meat products was also determined. Bacterial counts from raw game meat ranged from 2.37 to 5.37 log cfu/g. Counts as high as 6.16 log cfu/g were enumerated from retail products. Aerobic plate counts (APC) from ≤ 2.62 log cfu/cm2 to ≤ 6.3log cfu/cm2 were enumerated from surfaces, hands and equipment during production. Results highlighted the inefficiency of cleaning procedures and revealed that contaminated meat can allow for bacterial contamination. To determine if muscle pH influences colour stability and microbial spoilage of game meat, normal (n=6) and dark, firm and dry (DFD) (n=6) black wildebeest Longissimus thoracis et lumborum (LTL) muscles were studied. pH affected colour, as initial (day 0) L*,a*,b*,C* and Hab values from Normal pH samples were significantly higher than values reported for DFD samples. Initial APC and Enterobacteriaceae counts from samples with Normal pH were not significantly different from counts reported for DFD samples. Initial contamination was linked to the harvesting and slaughter process. Further refrigeration (5±1ºC) for 12 days in an aerobic environment and analyses of samples every third day revealed that pH did not affect lightness and brownness as L* and b* values for DFD samples did not significantly differ overtime, the same trend was seen for samples with Normal pH. Normal pH samples showed a significant increase in a* and C* values until day 12, whilst Hab values decreased until the 12th day. The same trend was seen for a* and C* values for DFD samples until the 9th day as on the 12th day values increased. Similarly, Hab values for DFD samples decreased until the 9th day, then increased on the 12th day. Using the microbial spoilage limit of 6 log cfu/g, it was seen that DFD meat reached this limit earlier than samples with Normal pH. Overall, the study provides baseline information on the microbiological quality of game meat harvested in South Africa and slaughtered at a South African abattoir. / AFRIKAANSE OPSOMMING: Die plaaslike en internasionale verbruik van wildsvleis en wildsvleisprodukte is aan’t toeneem. Hierdie toename in verbuik vereis navorsing wat gefokus is op die mikrobiese kwaliteit van wildsvleis. Die oes-en slagproses van springbok karkasse het die teenwoordigheid van bakteriese kontaminasie aan die lig gebring. Monsters geneem met ʼn depper na afslag van karkasse het ʼn teenwoordigheid van Escherichia coli (E. coli) getoon. Springbok karkasse wat getoets is na verkoeling het hoë vlakke van die aërobiese bakterium Clostridium spp. en van melksuurbakterieë getoon. In teenstelling hiermee is getalle laer rondom die ontweidings insnyding. Bakteriese kontaminasie was gekoppel aan swak higiëne gedurende die oes- en slagproses. Hierdie resultate het ʼn ondersoek van die slagproses aangemoedig. Om die impak van die slagproses op die mikrobiese kwaliteit van wildskarkasse te evalueer, is monsters regdeur geneem van swartwildebees (Connochaetes gnou). Getalle van aërobiese bakterieë, Enterobacteriaceae, en E. coli was bepaal op vel monsters voor afslag; getalle het gewissel tussen 0.92 en 7.84 log cve/g. Getalle van bakterieë na afslag het gewissel tussen 0.93 en 6.12 log cfu/g, en het verder afgeneem na verkoeling. Clostridum spp. het toegeneem na afslag, maar statistiese analises het geen beduidende verskille getoon nie. Monsters het negatief getoets vir Salmonella spp. Die resultate toon aan dat bakteriese kontaminasie wel plaasvind gedurende die slagproses. Die higiëniese status gedurende die produksie van wildsvleis is ook vasgestel. Bakteriegetalle van rou wildsvleis het gewissel tussen 2.37 log cve/g en 5.37 log cve/g. Getalle van handelsprodukte het getalle getoon van soveel as 6.16 log cve/g. Aërobiese plaat telling tussen ≤2.62 cve/cm2 en ≤ 6.3log cve/cm2 is vasgestel vanaf oppervlakte, hande en toerusting gedurende produksie. Resultate beklemtoon die ondoeltreffendheid van skoonmaakprosedures en wys dat aangetaste vleis bakteriese kontaminasie kan toelaat. Om te bepaal of die kleurstabiliteit en mikrobiese bederf van wildsvleis geaffekteer word deur spiere se pH, is normale (n=6) en donker, ferm, en droë (DFD) (n=6) Longissimus thoracis et lumborum (LTL) spiere van die swartwildebees bestudeer. Kleur was geaffekteer deur vleis pH, siende dat die aanvanklike waardes (dag 0) vir L*, a*, b*, C* en Hab aansienlik hoër was vir monsters met normale pH as DFD monsters. Aanvanklike getalle van aërobiese plaat telling en Enterobacteriaceae telling van monsters met Normale pH het nie beduidend verskil van DFD monsters nie. Aanvanklike besmetting was gekoppel aan die oes- en slagproses. Verdere verkoeling (5±1ºC) vir 12 dae in ʼn aërobiese omgewing en analise van monsters wys dat pH nie ligtheid en bruinheid affekteer nie; waardes vir L* en b* vir DFD monsters het nie beduidend verskil oor tyd nie. Dieselfde geld vir monsters met Normale pH. Monsters met Normale pH het ʼn beduidende toename in a* en C* getoon tot en met dag 12, terwyl waardes vir Hab afgeneem het tot en met dag 12. Dieselfde patroon is waargeneem by waardes vir a* en C* vir DFD monsters tot en met dag 9, terwyl dit toegeneem het op die 12de dag. Soortgelyk het Hab waardes vir DFD monsters afgeneem tot n met dag 9, en toegeneem op die 12de dag. Dit is ook gevind dat DFD vleis die limiet vir mikrobiese bederf (6 log cve/g) vroeër bereik as monsters met Normale pH. Die studie voorsien basis inligting oor die mikrobiese kwaliteit van wildsvleis wat geoes is in Suid Afrika, en geslag is by Suid Afrikaanse slagpale.

Arsenic is a naturally occurring toxic metal and its presence in food composites could be a potential risk to the health of both humans and animals. Arseniccontaminated groundwater is often used for food and animal consumption, irrigation of soils, which could potentially lead to arsenic entering the human food chain. Its side effects include multiple organ damage, cancers, heart disease, diabetes mellitus, hypertension, lung disease and peripheral vascular disease. Research investigations, epidemiologic surveys and total diet studies (market baskets) provide datasets, information and knowledge on arsenic content in foods. The determination of the concentration of arsenic in rice varieties is an active area of research. With the increasing capability to measure the concentration of arsenic in foods, there are volumes of varied and continuously generated datasets on arsenic in food groups. Visual analytics, which integrates techniques from information visualization and computational data analysis via interactive visual interfaces, presents an approach to enable data on arsenic concentrations to be visually represented. The goal of this doctoral research in Environmental Science is to address the need to provide visual analytical decision support tools on arsenic content in various foods with special emphasis on rice. The hypothesis of this doctoral thesis research is that software enabled visual representation and user interaction facilitated by visual interfaces will help discover hidden relationships between arsenic content and food categories. The specific objectives investigated were: (1) Provide insightful visual analytic views of compiled data on arsenic in food categories; (2) Categorize table ready foods by arsenic content; (3) Compare arsenic content in rice product categories and (4) Identify informative sentences on arsenic concentrations in rice. The overall research method is secondary data analyses using visual analytics techniques implemented through Tableau Software. Several datasets were utilized to conduct visual analytical representations of data on arsenic concentrations in foods. These consisted of (i) arsenic concentrations in 459 crop samples; (ii) arsenic concentrations in 328 table ready foods from multi-year total diet studies; (iii) estimates of daily inorganic arsenic intake for 49 food groups from multicountry total diet studies; (iv) arsenic content in rice product categories for 193 samples of rice and rice products; (v) 758 sentences extracted from PubMed abstracts on arsenic in rice. Several key insights were made in this doctoral research. The concentration of inorganic arsenic in instant rice was lower than those of other rice types. The concentration of Dimethylarsinic Acid (DMA) in wild rice, an aquatic grass, was notably lower than rice varieties (e.g. 0.0099 ppm versus 0.182 for a long grain white rice). The categorization of 328 table ready foods into 12 categories enhances the communication on arsenic concentrations. Outlier concentration of arsenic in rice were observed in views constructed for integrating data from four total diet studies. The 193 rice samples were grouped into two groups using a cut-off level of 3 mcg of inorganic arsenic per serving. The visual analytics views constructed allow users to specify cut-off levels desired. A total of 86 sentences from 53 PubMed abstracts were identified as informative for arsenic concentrations. The sentences enabled literature curation for arsenic concentration and additional supporting information such as location of the research. An informative sentence provided global “normal” range of 0.08 to 0.20 mg/kg for arsenic in rice. A visual analytics resource developed was a dashboard that facilitates the interaction with text and a connection to the knowledge base of the PubMed literature database. The research reported provides a foundation for additional investigations on visual analytics of data on arsenic concentrations in foods. Considering the massive and complex data associated with contaminants in foods, the development of visual analytics tools are needed to facilitate diverse human cognitive tasks. Visual analytics tools can provide integrated automated analysis; interaction with data; and data visualization critically needed to enhance decision making. Stakeholders that would benefit include consumers; food and health safety personnel; farmers; and food producers. Arsenic content of baby foods warrants attention because of the early life exposures that could have life time adverse health consequences. The action of microorganisms in the soil is associated with availability of arsenic species for uptake by plants. Genomic data on microbial communities presents wealth of data to identify mitigation strategies for arsenic uptake by plants. Arsenic metabolism pathways encoded in microbial genomes warrants further research. Visual analytics tasks could facilitate the discovery of biological processes for mitigating arsenic uptake from soil. The increasing availability of central resources on data from total diet studies and research investigations presents a need for personnel with diverse levels of skills in data management and analysis. Training workshops and courses on the foundations and applications of visual analytics can contribute to global workforce development in food safety and environmental health. Research investigations could determine learning gains accomplished through hardware and software for visual analytics. Finally, there is need to develop and evaluate informatics tools that have visual analytics capabilities in the domain of contaminants in foods. / Environmental Sciences / P. Phil. (Environmental Science)

Submitted by Alexandre Sousa (alexandre.sousa@incqs.fiocruz.br) on 2015-04-07T12:09:12Z No. of bitstreams: 1 Dissertação_Serodio.pdf: 586656 bytes, checksum: 807186c9c0240e06062add7281e114c6 (MD5) / Approved for entry into archive by Alexandre Sousa (alexandre.sousa@incqs.fiocruz.br) on 2015-04-07T12:09:28Z (GMT) No. of bitstreams: 1 Dissertação_Serodio.pdf: 586656 bytes, checksum: 807186c9c0240e06062add7281e114c6 (MD5) / Approved for entry into archive by Alexandre Sousa (alexandre.sousa@incqs.fiocruz.br) on 2015-04-07T12:09:39Z (GMT) No. of bitstreams: 1 Dissertação_Serodio.pdf: 586656 bytes, checksum: 807186c9c0240e06062add7281e114c6 (MD5) / Made available in DSpace on 2015-04-07T12:09:39Z (GMT). No. of bitstreams: 1 Dissertação_Serodio.pdf: 586656 bytes, checksum: 807186c9c0240e06062add7281e114c6 (MD5) Previous issue date: 2014 / Fundação Oswaldo Cruz. Instituto Nacional de Controle de Qualidade em Saúde / A ampla diversidade do uso dos agrotóxicos pode causar danos ambientais e à saúde humana que muitas vezes, são irreversíveis, ou cujas ações mitigatórias podem ser econômicas, políticas ou socialmente inviáveis. Segundo a Organização Mundial da Saúde, os agrotóxicos são potencialmente tóxicos para outros organismos, incluindo seres humanos, portanto necessitam que sejam descartados apropriadamente e utilizados de maneira segura. Os agrotóxicos são avaliados previamente à produção, à exportação, à importação, à comercialização e ao uso sendo por lei, obrigatório o registro desses produtos com a avaliação dos órgãos federais responsáveis pelos setores de saúde, meio ambiente e agricultura. Tal avaliação visa identificar potenciais danos pelo uso dessas substâncias, com o objetivo de estabelecer proibições, restrições e recomendações para o uso de agrotóxicos, prevenindo e dimensionando os perigos à saúde humana e meio ambiente. A proibição de agrotóxicos que provoquem tais danos deve ser feita a partir de informações dos achados científicos e sua relevância para a exposição humana. A relevância dos estudos experimentais que utilizam sistemas in vitro ou in vivo, se dá a partir da elucidação dos modos de ação tóxica do agrotóxico. O presente trabalho teve como objetivo a reavaliação toxicológica do ingrediente ativo metano arseniato ácido monossódico (MSMA) através de um levantamento bibliográfico criterioso. O intuito é o banimento do MSMA visto que ele quando é metabolizado no solo se biotransforma em espécies arsenicais extremamente tóxicas. Estudos demonstraram que os metabólitos do MSMA que são: As V,As III, MMA e DMAque causam efeitos carcinogênico, teratogênico entre outros que são proibitivos ao seu uso. Este trabalho priorizou os achados mais relevantes para a saúde pública visando o risco do uso de um agrotóxico que causa efeito deletério. / The wide diversity of the use of pesticides can damage the environment and human health, which often are irreversible or whose mitigating actions can be economic, political or socially unviable. According to the World Health Organization, pesticides are potentially toxic to other organisms, including humans, and therefore need to be safely used and properly disposed. Pesticides are evaluated prior to its production, export, import, marketing and use having by law its required registration assessed by the federal agencies responsible for the health, environment and agriculture. This evaluation aims to identify potential damage due to the use of these substances, establishing prohibitions, restrictions and recommendations for their use, preventing and measuring the dangers to human health and the environment. The banishment of pesticides that cause such damage should be basedon information from scientific findings and their relevance to human exposure. The importance of experimental studies using in vitro systems or in vivo, anchors in the elucidation of toxic action mechanisms of pesticides. The present study aimed a toxicological re-evaluation of the active ingredient monosodium methane arsenate acid (MSMA) through a careful literature survey and its official ban, since it is metabolized when the soil biotransforms in extremely toxic arsenic species. Studies have shown that the metabolites of MSMA: AsV, AsIII, MMA and DMA cause carcinogenic, teratogenic and others effects that make their use prohibitive. This work prioritized the most relevant findings for public health about the risk of using a pesticide that causes deleterious effect.

Submitted by JESSELEINE CRISTINE MONTEIRO DA SILVA null (jesse_cris@hotmail.com) on 2017-09-20T20:56:09Z No. of bitstreams: 1 Dissertação final Jesseleine.pdf: 1516064 bytes, checksum: 56e50bf83bbdb465716b33121512f13f (MD5) / Approved for entry into archive by Monique Sasaki (sayumi_sasaki@hotmail.com) on 2017-09-27T17:48:51Z (GMT) No. of bitstreams: 1 silva_jcm_me_araiq.pdf: 1516064 bytes, checksum: 56e50bf83bbdb465716b33121512f13f (MD5) / Made available in DSpace on 2017-09-27T17:48:51Z (GMT). No. of bitstreams: 1 silva_jcm_me_araiq.pdf: 1516064 bytes, checksum: 56e50bf83bbdb465716b33121512f13f (MD5) Previous issue date: 2017-09-06 / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Devido ao crescente aumento de doenças transmitidas por alimentos, a segurança microbiológica se torna uma questão de saúde pública pelas suas características de endemicidade, alta morbidade e pela dificuldade da adoção de medidas de controle desses microrganismos. Diante deste fato, o objetivo deste trabalho foi sintetizar e caracterizar os análogos peptídicos LeuB e LeuC-1 derivados de bacteriocinas naturais denominadas Leucocinas. Os peptídeos foram sintetizados manualmente pelo método de síntese em fase sólida, submetidos à desproteção total e clivagem, com liberação dos peptídeos brutos. Foram realizadas as análises comparativas usando HPLC e ESI-MS, e com os respectivos peptídeos puros foram feitos os ensaios antimicrobiano, enzimático, permeabilização, antioxidante, hemolítico e de espectroscopia de dicroísmo circular. Com isso, observou-se que o método de síntese dos análogos foi adequado e o processo de purificação possibilitou a obtenção dos peptídeos com alto grau de pureza. O peso molecular teórico dos peptídeos foi confirmado por espectrometria de massas. O LeuB apresentou uma maior capacidade em inibir o crescimento de Escherichia coli O157:H7 e em Salmonella sorovar Typhimurium, enquanto que LeuC-1 apresentou efeito de inibição de crescimento de Listeria monocytogenes e também de S. sorovar Typhimurium. É importante destacar que todas essas bactérias são de interesse na área de alimentos, já que são as causadoras da maioria dos casos de infecção alimentar. O ensaio de inibição enzimática com DNA girase e Topoisomerase IV mostrou que apenas o peptídeo LeuB possui capacidade de inibição destas enzimas bacterianas, sugerindo um possível mecanismo de ação deste derivado de Leucocina. Este peptídeo também apresentou a capacidade de permeabilizar miméticos de membrana bacteriana composto de POPC/POPG (75/25). Por sua vez, o peptídeo LeuC-1 não apresentou capacidade significativa de inibição da atividade das enzimas DNA girase e Topoisomerase IV e também não apresentou capacidade de permeabilização de miméticos de membrana. Porém, LeuC-1 apresentou uma boa atividade antioxidante, obtida pelo método de ABTS. Ambos os peptídeos apresentaram baixa toxicidade em eritrócitos, comprovadas pelos ensaios hemolíticos. Estruturalmente, os peptídeos LeuB e LeuC-1 tendem a se estruturar em α-hélice. Assim sendo, este trabalho possibilitou a obtenção de dois peptídeos com potencial de aplicação como conservantes alimentares a partir de mecanismos distintos de ação sem apresentar citotoxicidade para células vermelhas do sangue. LeuB possui uma suposta atuação como inibidor de topoisomerases bacterianas e capacidade de permeabilização de miméticos de membrana. LeuC-1 possivelmente atua em diferentes vias metabólicas da bactéria, porém ainda não foi possível elucidar o mecanismo alvo deste mimético peptídico. / Due to the increase of foodborne diseases, microbiological safety becomes a public health issue due to its characteristics of endemicity, high morbidity and the difficulty of adopting control measures of these microorganisms. In view of this fact, the objective of this work was to synthesize and characterize LeuB and LeuC-1 peptidics analogues derived from natural bacteriocins called Leucocins. The peptides were synthesized manually by the solid phase synthesis method, subjected to total deprotection and cleavage, with release of the crude peptides. Comparative analyzes were performed using HPLC and ESI-MS, and with the respective pure peptides the antimicrobial, enzymatic, permeabilization, antioxidant, hemolytic and circular dichroism spectroscopy. With this, it was observed that the method of synthesis of the analogs was adequate and the purification process allowed to obtain the peptides with high purity. The theoretical molecular weight of the peptides was confirmed by mass spectrometry. LeuB showed a greater capacity to inhibit the growth of Escherichia coli O157: H7 and Salmonella serovar Typhimurium, whereas LeuC-1 presented inhibition effect of growth of Listeria monocytogenes and also of S. serovar Typhimurium. It is important to highlight that all these bacteria are interesting in the area of food, since they are the cause of most cases of food infection. The enzyme inhibition assay with DNA gyrase and Topoisomerase IV showed that only the LeuB peptide has the ability to inhibit these bacterial enzymes, suggesting a possible mechanism of action of this Leucocin derivative. This peptide also showed the ability to permeabilize bacterial membrane mimetics composed of POPC/POPG (75/25). On the other hand, the LeuC- 1 peptide did not present significant capacity to inhibit the activity of the enzymes DNA gyrase and Topoisomerase IV and also did not present permeabilization capacity of membrane mimetics. However, LeuC-1 presented a good antioxidant activity, obtained by the ABTS method. Both peptides had low erythrocyte toxicity, as demonstrated by hemolytic assays. Structurally, the LeuB and LeuC-1 peptides tend to be α-helix structured. Therefore, this work enabled two peptides with application potential as food preservatives to be obtained from distinct mechanisms of action without presenting red blood cell cytotoxicity. LeuB has a supposed action as inhibitor of bacterial topoisomerases and permeabilization capacity of membrane mimetics. LeuC-1 possibly acts on different metabolic pathways of the bacterium, but it has not yet been possible to elucidate the target mechanism of this peptidic mimetic. / CNPq: 150928/2015-7

O leite é um dos alimentos mais completos da natureza e sua importância é baseada em seu elevado valor nutritivo, como riqueza de proteínas, vitaminas, gordura, sais minerais e a alta digestibilidade. Esses fatores são relevantes para considerá-lo um excelente meio de cultura para a maioria dos microrganismos. A pasteurização é necessária e tem a finalidade de eliminar os microrganismos patogênicos, além de diminuir ao máximo o número de microrganismos em geral, mas alguns deles ainda podem sobreviver ao tratamento térmico aplicado. O objetivo do presente trabalho foi avaliar a condição microbiológica e a eficácia da pasteurização industrial do leite tipos A, B, C e as condições microbiológicas do leite cru, através da enumeração de bactérias aeróbias mesófilas, termófilas e psicrotróficas, Staphylococcus coagulase positiva, determinação do Número Mais Provável (NMP) de coliformes totais e fecais e pesquisa de Salmonella spp. em 30 amostras de leite comercializado no município de Piracicaba- SP. Com base na legislação do DIPOA (Brasil, 2002) os resultados mostraram que 44,4% das amostras de leite tipo A apresentaram-se fora do padrão microbiológico para aeróbios mesófilos. Para coliformes totais e fecais, o mesmo leite apresentou 66,7% e 55,6% das amostras respectivamente, em desacordo com a legislação vigente. No leite tipo B, nenhuma amostra esteve fora do padrão microbiológico em relação a aeróbios mesófilos. Já para coliformes totais e fecais, 33,3% das amostras estiveram em desacordo com a legislação em vigor. O leite tipo C foi o que apresentou resultado mais satisfatório, pois ao contrário do que se esperava, nenhuma amostra esteve fora do padrão em relação às análises microbiológicas realizadas no presente trabalho. O leite cru analisado apresentou valores elevados para todas as análises microbiológicas realizadas. Não foram encontradas amostras contaminadas com Salmonella spp. em todas as amostras de leite analisadas. O maior valor encontrado para Staphylococcus coagulase positiva foi 1,1 x 102 UFC/mL, portanto, longe da dose infectiva, mas esse fato não deixa de ser preocupante, já que o leite é um ótimo substrato para bactérias. Após a pasteurização (62,8ºC/30’) realizada no Laboratório de Microbiologia de Alimentos, do Departamento de Agroindústria, Alimentos e Nutrição, da ESALQ, todas as amostras novamente analisadas para os parâmetros microbiológicos citados, se mostraram em acordo com a legislação nacional vigente (Brasil, 2002). Os resultados encontrados na presente pesquisa podem ser indicativos de prováveis falhas do binômio tempo/temperatura durante a pasteurização industrial; matéria-prima excessivamente contaminada; higienização e sanificação deficientes das linhas de produção ou contaminação pós-pasteurização. / Milk is one of the most valuable of all foods in nature and its importance is based on its high nutritive value. Milk is rich in proteins, vitamins, fat, mineral salts, and high digestibility. These factors are relevant for it to be considered an excellent culture medium for most microorganisms. Pasteurization is necessary and its main purpose is to eliminate pathogenic microorganisms, in addition to reducing to the maximum the number of microorganisms in general. However, some of them may still outlive the thermal treatment applied. The aim of this project was to evaluate the microbiological conditions and efficiency of the industrial pasteurization of type-A,-B and-C milk, and the microbiological conditions of raw milk, through the number of mesophillic, thermophilic and psychrotrophic aerobe bacteria, Staphylococcus positive coagulase, through the determination of the Most Probable Number (MPN) of total and fecal coliforms, and also through the research on Salmonella spp. in 30 samples of milk commercialized in Piracicaba-SP. Based on DIPOA legislation (Brazil, 2002), the results showed that 44.4% of the type-A milk samples did not meet the microbiological standard for mesophillic aerobe for total and fecal coliforms, 66.7% and 55.6% of the same milk samples, respectively, were not in accordance with the present legislation. For the type-B milk, no sample failed to meet the microbiological standard in relation to mesophillic aerobes. However, for total and fecal coliforms, 33.3% of the samples were in disagreement with the present legislation. Type-C milk was the one presenting the best result. All samples were in accordance with the microbiological analysis performed in this work. The raw milk examined showed high values for all microbiological analyses. No infected samples with Salmonella spp. were found in the analyzed milk samples. The greatest value found for Staphylococcus positive coagulase was 1.1 x 102 UFC/mL, thus, far from the infective dose, although somewhat concearning since milk is considered na excellent environment for the infestation of bacteria. After the pasteurization (62.8ºC/30’) performed in the Food Microbiology Laboratory, of the Agroindustry, Foods and Nutrition Department, ESALQ/USP, all samples that analyzed again, based on the microbiological parameters mentioned above, met the national legislation in force (Brazil, 2002). The results found in the research may be an indication of probable failures regarding time/temperature during the industrial pasteurization; infected raw-material; defecient sanitation of product lines or post-pasteurization contamination.

Doctorat en Sciences / info:eu-repo/semantics/nonPublished

Listeria monocytogenes é de grande preocupação para a sande pública e para indústrias de alimentos, pois pode causar meningite, septicemia e aborto. Trabalhos desenvolvidos em diversos países, bem como no Brasil, indicam que a presenca de L. monocytogenes nas indústrias processadoras de pescado é freqüente. Corn a finalidade de monitorar a contaminação por L. monocytogenes em plantas processadoras de alimentos e identificar as fontes de contaminação do produto têm-se empregado técnicas de biologia molecular. Dentre elas cabe destacar a PFGE (pulsed field gel electrophoresis - eletroforese em gel de campo pulsado), técnica baseada em macrorestrição genômica, através da utilização de uma enzima de restrição de baixa freqüncia de corte, como a SmaI , AscI e ApaI, seguido de uma eletroforese em gel em campo pulsado. Neste estudo foram utilizadas 181 cepas de L. monocytogenes isoladas a partir de amostras de salmão \"gravlax\" colhidas nas diferentes etapas de processamento, além de amostras de manipuladores, ambientes e utensílios, coletadas em uma usina de processamento industrial a fim de verificar a diversidade antigênica e genética. Estas cepas foram sorogrupadas corn os antissoros 0 tipo 1 e 4 e subtipadas após PFGE seguindo o protocolo preconizado pelo Center for Disease Control and Prevention e empregando as enzimas ApaI e AscI. Das cepas, 132 (72,9%) pertenceram ao sorogrupo 1 e 49 (27,1%) ao sorogrupo 4. Corn as enzimas ApaI e AscI foram obtidos, respectivamente, 30 e 28 perfis de macrorestriçãoo diferentes. Os perfis de ambas as enzimas foram combinados obtendo-se 60 perfis combinados de macrorestrição. A combinação dos perfis de macrorestrição à sorologia permitiu separar as cepas em 8 subtipos e a distribuição destes subtipos na linha de processamento foi avaliada. Notou-se que um mesmo perfil de macrorestrição foi encontrado no salmão desde o início do processamento até o produto final, já embalado, utensílios e nas mãos de manipuladores. Também verificou-se que alguns perfis estavam adaptados ao ambiente de processamento e utensílios não sendo transmitidos ao produto. Na planta em questão há necessidade de aplicação inicialmente de Boas Práticas de Fabricação (BPF) e posteriormente Análise de Perigos e Pontos Críticos de Controle (HACCP). / Listeria monocytogenes is a great concern for the public health and for the food industry, because it can cause serious illness such as meningitis, septicemia and abortion. Lots of studies in many countries, including Brazil, point to the frequent presence of L. monocytogenes in the fish industries. Molecular biology techniques have been applied to trace the contamination of L. monocytogenes and identify the sources of contamination of the food processing lines. One of them is the PFGE (pulsed-field gel electrophoresis), which is based on a genomic macrorestriction with low frequency restriction enzymes (SmaI, AscI and Apal), followed by a pulsed electrophoresis. 181 strains of L. monocytogenes were isolated from \"gravlax\" salmon processing line in several stages of the industrial processing, handworkers, environmental and food contact surfaces samples and the antigenic and genetic diversity were evaluated. They were serogrouped with antiserum 0 type 1 and 4, and subtyped using PFGE according to Center for Disease Control and Prevention protocol using the enzymes ApaI and AscI. 132 strains (72,9%) belonged to serogroup 1, 49 (27,1%) to serogroup 4. Visual comparison for macrorestriction patterns revealed 30 distinct ApaI restriction endonuclease digestion profiles (REDP) and 28 AscI REDP. When the composite profile from both enzymes was generated, there were 60 profiles. The combined macrorestriction profiles were joined to the serology resulting in 8 subtypes and the subtypes distribution along the processing line was evaluated. It\'s interesting to note that one specific profile found in the raw material was also found in different processing steps up to the final product (readyto-eat \"gravlax\" salmon) and also in food contact and non food contac surfaces, as well as food handlers. Some profiles were well adapted to the processing environment and were not found in fish. Our results strongly suggest that this industry needs to apply the Good (GMP) and Analysis of Hazards and Critical Points of Control (HACCP) to produce safer products.

O presente trabalho teve por objetivos determinar possíveis fontes de contaminação dos suínos por Salmonellanas granjas de criação; verificar o comportamento de Salmonellana cadeia produtiva de suínos (do nascimento à terminação e ao longo das etapas de transporte, descanso, abate e frigorificação); correlacionar os sorotipos de Salmonellasp. isolados durante as etapas de criação e pré-abate com aqueles oriundos do abate e frigorificação das carcaças; caracterizar o perfil de resistência de Salmonellasp. utilizando como ferramenta para diferenciação dos sorotipos encontrados, verificar a eficiência de metodologias de isolamento de Salmonella. Desde o nascimento até o abate, a cada 10 dias, foram coletadas amostras de fezes, de ambiente, de ração do cocho, de ração da fábrica e estoque, da água de beber e água de abastecimento em 6 lotes de suínos provenientes de 03 granjas distintas, (2 lotes de cada) localizadas na região oeste do Estado do PR. Destes mesmos lotes, foi acompanhado o pré-abate e abate através da coleta de amostras em 12 pontos ao longo do fluxograma de abate. Durante o confinamento, independente do lote, do ponto ou da data foram isolados 9 sorotipos diferentes de Salmonella(S. 4,5, S. 4,5,12:i:-, S. 4,5,12:i:1,2, S. Agona, S. Derby, S. Mbandaka, S. Panama, S. Poona, S. Typhimurium) e uma cepa não tipável (S. enterica subsp. enterica cepa rugosa). Das amostras coletadas durante o pré-abate e o abate também foram identificadas 9 sorotipos, 3 dos quais não haviam sido isolados nas granjas (S. Give, S. Meleagridis e S. Worthington), sendo que não foram detectados os sorotipos S. Derby, S. 4,5,12:-:1,2 e S. Poona. Foi elevado o percentual de cepas de Salmonellaresistentes à pelo menos um antimicrobiano (93,7%) sendo que 62,9% foram resistentes a mais de um. Pelos resultados encontrados pode-se chegar as seguintes conclusões: a etapa de terminação foi aquela com maior importância para o isolamento de Salmonellasp. durante a criação; a existência de cepas multi-resistentes a antibióticos deve ser considerada como risco à saúde pública; o transporte e descanso dos animais nas pocilgas do matadouro mostraram ser etapas importantes para o aumento da excreção de Salmonellasp. pelos suínos portadores; A contaminação existente nas granjas e em algumas etapas do início do processo de abate não se refletiu nas carcaças abatidas e frigorificadas, em virtude de um processamento tecnológico de abate bem executado; o pré-enriquecimento mostrou ser uma etapa essencial para o isolamento de Salmonellaa partir de fezes e o método alternativo utilizado mostrou ser eficiente como um método de triagem para a identificação de Salmonellasp. das amostras analisadas. / The objective of the present trial was to correlate the serotypes of Salmonellasp. isolated during breeding and pre-slaughter with those isolated from slaughter and cooling of carcasses, according to the \"from farm to table\" concept. From birth to slaughter, every 10 days, samples of feces, the environment of the facility, feed leftovers, feed upon arrival and feed in stock, drinking water and supply water were collected from 6 groups of swine of 3 different breeding units (2 groups of each) located in the west region of the state of Parana, Brazil. The same groups were sampled during pre-slaughter and slaughter in 12 points throughout the slaughter procedure. During confinement, no matter the group, sampling point or date, 9 different Salmonellaserotypes were isolated (S. 4,5, S. 4,5,12:i:-, S. 4,5,12:i:1,2, S. Agona, S. Derby, S. Mbandaka, S. Panama, S. Poona, S. Typhimurium), besides one non-typed strain (rough S. enterica subsp. enterica). From the samples collected during pre-slaughter and slaughter, 9 serotypes were identified, of which 3 had not been isolated in the breeding facilities (S. Give, S. Meleagridis and S. Worthington). Serotypes S. Derby, S. 4,5,12:-:1,2 and S. Poona were not identified. A large percentile of Salmonellastrains was resistant to at least one antimicrobial compound (93.7%), and 62.9% were resistant to more than one. The existence of multiresistant strains should be considered as a public health problem. It was observed that feces presented the greatest epidemiological importance in the dissemination of Salmonellain all breeding stages, and confinement-finishing units were the most important sites of Salmonellasp isolation; transport and rest of the animals in the slaughter pens were important stages in the increase of the Salmonellashedding by carrier animals; there was no direct correlation between the contamination of the breeding facility and positive results obtained in the slaughter, as assessed by carcass contamination.