Submitted by Marco Antônio de Ramos Chagas (mchagas@ufv.br) on 2017-05-04T14:05:26Z No. of bitstreams: 1 texto completo.pdf: 884943 bytes, checksum: 6d04675a5504c3d18ce512015afd015c (MD5) / Made available in DSpace on 2017-05-04T14:05:26Z (GMT). No. of bitstreams: 1 texto completo.pdf: 884943 bytes, checksum: 6d04675a5504c3d18ce512015afd015c (MD5) Previous issue date: 2005-04-26 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / O Brasil é o principal produtor de feijão no mundo, com mais de três milhões de toneladas produzidas anualmente. Entretanto, esta produtividade poderia ser maior. Diversos fatores têm impedido que a produção do país aumente, entre eles, a ocorrência de doenças durante o ciclo da cultura tem papel central. Mais de duzentos patógenos já foram relatados ocorrendo em feijoeiro, embora apenas uma dúzia tenha sido relatada como importante. No Brasil, o crestamento bacteriano, a mancha angular, a ferrugem e a antracnose são as principais doenças da parte aérea da cultura, podendo reduzir a produção em até 70%. Atualmente, as medidas de controle adotadas têm sido relativamente eficientes devido à alta variabilidade dos patógenos no campo e pequeno número de genes de resistência disponíveis e o plantio das mesmas cultivares por ciclos sucessivos. Assim o controle biológico tem papel fundamental, quando inserido numa estratégia de manejo integrado de doenças. Dentre os organismos estudados para atuarem no biocontrole de doenças, as bactérias têm sido relatadas como capazes de inibir outros microrganismos por meio de mecanismos de antibiose, parasitismo, competição e indução de resistência. O presente trabalho teve como objetivos selecionar bactérias do filoplano de feijoeiro como agentes de biocontrole de doenças da parte aérea da cultura, baseado em estratégias de seleção conjuntas in vitro e in vivo, determinar que mecanismos de controle poderiam estar envolvidos, se os isolados obtidos estariam atuando como promotores de crescimento ou indutores de resistência e testar a efetividade dos isolados em campo, em diferentes cultivares e em idades diferentes da cultura, contra os patógenos desafiantes, Colletotrichum lindemuthianum, Erysiphe polygoni, Phaeoisariopsis griseola, Pseudomonas viridiflava, Uromyces appendiculatus e Xanthomonas axonopodis pv. phaseoli. Dos 500 isolados obtidos, três foram mais promissores, quanto ao controle das doenças em casa de vegetação, reduzindo a severidade das doenças quando comparados ao tratamento com água, sendo dois identificados por sequenciamento do RNA16S como Bacillus cereus (UFV-75 e UFV-172) e o terceiro, por análise de ácidos graxos, como Pseudomonas putida (UFV-108). Nos ensaios de antibiose in vitro, verificou-se que a bactéria Bacillus cereus (UFV-75) foi capaz de inibir, seja a germinação de conídios ou o crescimento micelial de fungos ou o crescimento de bactérias, todos os patógenos testados. Demonstrou-se em ensaio in vitro que B. cereus (UFV-75) foi produtor de sideróforos, compostos voláteis, bacteriocinas e da enzima quitinase. As bactérias B. cereus (UFV-172) e P. putida (UFV-108) também produziram sideróforos, embora o isolado UFV-172 não tenha inibido o crescimento de nenhum dos patógenos in vitro. Nos ensaios de campo, UFV-75, UFV-108 e UFV-172, reduziram a severidade da mancha angular (P. griseola) e da ferrugem (U. appendiculatus). Nos ensaios de promoção de crescimento, o isolado UFV-74 (Pseudomonas putida) foi selecionado, por promover o aumento do tamanho das folhas e das plantas em casa de vegetação e também aumentar a produtividade no campo, quando pulverizado sobre as folhas ou via microbiolização de sementes. Nos testes com produtos comerciais, B. cereus (os dois isolados) e P. putida foram sensíveis apenas aos fungicidas Cupravit Azul Br e Manzat 800. Nos ensaios de amplitude de controle, onde se avaliou a eficiência de B. cereus (UFV-172 e UFV-75) e P. putida, em controlar: a) o crestamento bacteriano em plantas da cultivar Pérola com idades diferentes; b) a antracnose, o oídio, a mancha bacteriana e a mancha angular na cultivar Pérola e; c) em nove cultivares diferentes contra o crestamento bacteriano, todos os três isolados foram eficientes em controlar o crestamento bacteriano comum em plantas com idades até 60 dias após a emergência. Todos os isolados foram capazes de reduzir a severidade da antracnose, do oídio, da mancha bacteriana e da mancha angular, exceto nos ensaios de UFV-75 X oídio e todos os isolados foram eficientes em controlar o crestamento bacteriano, em todas as cultivares testadas, exceto nos ensaios de UFV-75 X ‘Vermelhinho’. Nos ensaios de sistemicidade, onde buscava-se determinar se B. cereus (UFV-172) poderia estar atuando como indutor de resistência sistêmica, o isolado inibiu os patógenos desafiantes X. a. pv. phaseoli e M. incognita, quando aplicado em região separada espacialmente da região que foram aplicados os patógenos, dando indícios de que o isolado pode estar agindo nas plantas de feijoeiro como indutor de resistência. Esse resultado é corroborado pelo ensaio de restrição de multiplicação de X. a. pv. phaseoli, quando o isolado foi aplicado quatro dias antes do patógeno e esse, quando infiltrado em folhas de feijoeiro, teve um desenvolvimento inferior ao de plantas não expostas ao isolado, sugerindo que mecanismos de sinalização que induzem a síntese de compostos que restringem o desenvolvimento dos patógenos são desencadeados nas plantas quando estas são expostas ao isolado e que esse sinal é sistêmico sendo translocado das folhas até as raízes. / Brazil is the most important bean producer in the world, producing around 3 million tons of grains per year. Nevertheless, the Brazilian productivity could be higher but several factors have been impeding increases in productivity such as the occurrence of multiple diseases during the plant life cycle. In this sense, more than two hundred pathogens are known to induce diseases in bean but only a few actually are important. Under Brazilian conditions, the bacterial blight, the angular leaf spot, the rust and the anthracnose are the most important bean phylloplane diseases and may account for losses up to 70% if environmental conditions favor disease occurrence. Nowadays, the majority of disease control procedures lack efficiency due to inefficiency of available recommended chemicals in the field, the high level of variability in pathogen populations as well as the few resistance genes detected and manipulated. Therefore, biological control assumes paramount importance when included in global procedures of disease integrated management. Among focused microorganisms as potential biocontrol agents, bacteria have been exhaustively investigated and it is well known that they can act either by direct antibiosis or by inducing systemic resistance. This work had as main objective the selection of prokaryotic phylloplane residents that might act as agents of biological control for bean aerial part diseases, from a universe of 500 isolates obtained from healthy bean phylloplane. In this sense, the strategy of combining “in vitro” as “in vivo” procedures for the massal screen, the investigation on the nature of control mechanisms involved, specific tests to determine whether the observed biocontrol could be attributed to direct antibiosis or to induced resistance, specific assays to verify whether selected antagonists might act as growth promoters, field tests to investigate whether selected isolates would act as biocontrol agents when delivered to distinct bean cultivars, to plants in different phenology stages and against multiple pathogens (Xanthomonas axonopodis pv. phaseoli, Pseudomonas viridiflava, Uromyces appendiculatus, Colletotrichum lindemuthianum, Phaeoisariopsis griseola and Erysiphe polygoni) were studied. Out of the set of 500 isolates, three of them - UFV-75, UFV-172 (Bacillus cereus) and UFV-108 (Pseudomonas putida) – presented the best performance as biocontrol agents being able to reduce diseases severity up to 75% in greenhouse tests. In “in vitro” tests, isolate UFV-75 was able to inhibit growth of all pathogens as well as produce siderophores, volatile antimicrobial compounds, bacteriocins and chitinases. Isolates UFV-172 and UFV-108 also produced siderophores but the latter shown no “in vitro” activity against pathogens. In field trials, all three isolates showed efficiency for reducing severity of rust (U. appendiculatus) and angular leaf spot (P. griseola). In growth-promoting assays, an isolate distinct from the ones selected as biocontrol agents, namely UFV-74 (Pseudomonas putida), behaved as a good promoter, as detected by plant growth parameters evaluation like number of leaves, plant height and seed production, if delivered either by seed microbiolization or by spraying of propagules in the aerial part. Investigating the possibility of mixing the selected biocontrol agents and commercial products recommended in Brazil (registered pesticides, like fungicides, insecticides and herbicides) for the bean culture, it was found that all of them were sensitive only to the fungicides Cupravit Azul Br and Manzat 800. Using X. a. pv. phaseoli as challenging pathogen, it was found that all of them were able to low down disease severity but in the interaction isolate UFV-75 and “Vermelhinho” cultivar. All three selected isolates were able to promote the experimental biocontrol of bacterial blight under controlled conditions regardless plant age, up to 70 days after sowing. Using “Perola” as model cultivar, it was verified that protection brought about by the three isolates had character of multiplicity in the sense that this protection was effective against a broad range of pathogens like C. lindemuthianum, P. griseola P.viridiflava and Erysiphe polygoni. In protection sistemicity assays, the isolate UFV-172 provided protection against X. a. pv. phaseoli and Meloidogyne incognita even if delivered to locals remote from the site of inoculation. This constitutive sistemicity of protection indicates that isolate UFV-172 may promote biocontrol by inducing systemic resistance and the efficiency by which the antagonist restricted multiplication of a model pathogen (X. a. pv. phaseoli) in bean leaf tissue provided an additional evidence for that. / Tese importada do Alexandria

Consumer demand for fresher foods has necessitated the use of non-thermal technologies in processing milk. Two Dean Flow UV reactors (1/16" ID × 1/32" Thick & 1/8" ID × 1/32" Thick) were designed in the laboratory. The objective of this study was to examine the efficacy of designed UV reactors at four levels of Reynolds numbers (Re) on inactivation of Escherichia coli W 1485 cells and Bacillus cereus spores in raw cow milk (RCM), commercially processed skimmed cow milk (SCM) and raw soymilk (RSM). RCM, SCM and RSM were inoculated separately with E. coli W 1485 and B. cereus spores and were treated through the designed reactors for a residence time of 11.3 ± 0.1 s, equivalent to an UV dose of 0.05 J/ml in 1/16" ID reactor and 0.02 J/ml in 1/8" ID reactor. Four levels of Re were tested in the range of 181-1372. The influence of tube diameter (thickness of milk exposed to UV) and Re (indicator of turbulence) at constant residence time (11.3 ± 0.1 s) on inactivation of both the bacteria in both the UV reactors was analyzed using two-way ANOVA with proc GLM in SAS software. E. coli was inactivated to non-detectable levels (≥7.8 log10 CFU/ml) in SCM from the second level of Re (532.2) in 1/16" ID reactor. E. coli was also inactivated significantly (> 5logs) in RSM at the highest Re (1372) but this was not achieved in the case of RCM (712.7). Increasing the residence time to 14.2 s or greater (17 s) (equal to UV dose of 0.06 and 0.08 J/ml) inactivated E. coli cells to non-detectable levels in RCM using 1/16" ID reactor at the highest level of Re (712.7). Reduction of E. coli cells were in the range of 0.45-7.78 logs whereas B. cereus spores were in range of 1.06- 3.29 logs in all types of milk used in this study. The interaction effect of tube diameter and Re was statistically significant for E. coli cells in RCM, and SCM; B. cereus spores in SCM, and RSM (p < 0.05) whereas this was statistically non significant for E. coli cells in RSM and B. cereus spores in RCM (p > 0.05). Main effects of Reynolds number, and tube diameter were statistically significant (p < 0.05) on inactivation of B. cereus spores in RCM and E. coli cells in RSM. Inactivation efficiencies for both bacteria were higher in 1/16" ID reactor than 1/8" ID reactor. Using the 1/16" ID reactor at highest level of Re (RCM Re = 712.7, RSM Re = 1372), inactivation of standard plate count (SPC) present in RSM and RCM, and lipid oxidation during storage period (0, 1, 3 and 7 days) were measured. Inactivation of SPC in UV-treated RSM (3 logs) was lower than thermal pasteurization at 72°C for 20 s (7 logs). In case of RCM, the SPC was inactivated to 1.9 logs from 4.2 logs. Sensory evaluation (olfactory) of UV treated, untreated (milk passed through the 1/16" ID reactor while the UV lights turned off), and fresh RCM (control) suggested no change in flavor after treatment and upto 1 day after storage in refrigerated condition, but a perceivable change in the quality of UV treated and untreated cow milk were observed during the 3rd and 7th days when compared with fresh RCM (milked same day). RCM was treated with different UV dose levels (0.04, 0.05, 0.08, 0.12 and 0.16 J/ml) to examine the effect of UV light on malondialdehyde and other reactive substances using TBARS test kit. Reactive substances such as malondialdehyde content increased as the UV dose increased. The presence of malondialdehyde and other reactive substances were not significantly different (p < 0.05) in both thermal and UV-pasteurized soymilk; whereas these substances were found to be higher in UV-treated RCM after 7 days of storage than the untreated milk stored for 7 days at 4 °C and the fresh RCM. The designed reactors 1/16" ID and 1/8" ID reactors were useful to inactivate bacteria present in milk. But, the inactivation efficiency was more in 1/16" ID reactor than 1/8" ID reactor.

Orientador: Pilar Rodriguez de Massaguer / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-05T11:41:03Z (GMT). No. of bitstreams: 1 Sanches_CristianaDePaulaPacheco_D.pdf: 39491118 bytes, checksum: 1b9b67b564e9684ca74114092ff37150 (MD5) Previous issue date: 2005 / Resumo: O resumo poderá ser visualizado no texto completo da tese digital / Abstract: The abstract is available with the full electronic document / Doutorado / Doutor em Ciência de Alimentos

Submitted by Geyciane Santos (geyciane_thamires@hotmail.com) on 2015-07-03T15:08:24Z No. of bitstreams: 1 Dissertação - Marcos César Fernandes Pessoa.pdf: 6006738 bytes, checksum: 6bf14f8b1d3cb3d2908ac7e37e22c668 (MD5) / Approved for entry into archive by Divisão de Documentação/BC Biblioteca Central (ddbc@ufam.edu.br) on 2015-07-13T15:51:34Z (GMT) No. of bitstreams: 1 Dissertação - Marcos César Fernandes Pessoa.pdf: 6006738 bytes, checksum: 6bf14f8b1d3cb3d2908ac7e37e22c668 (MD5) / Approved for entry into archive by Divisão de Documentação/BC Biblioteca Central (ddbc@ufam.edu.br) on 2015-07-13T15:56:30Z (GMT) No. of bitstreams: 1 Dissertação - Marcos César Fernandes Pessoa.pdf: 6006738 bytes, checksum: 6bf14f8b1d3cb3d2908ac7e37e22c668 (MD5) / Made available in DSpace on 2015-07-13T15:56:30Z (GMT). No. of bitstreams: 1 Dissertação - Marcos César Fernandes Pessoa.pdf: 6006738 bytes, checksum: 6bf14f8b1d3cb3d2908ac7e37e22c668 (MD5) Previous issue date: 2009-07-15 / CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico / Bacillus thuringiensis is a Gram-positive bacterium commonly used in the tropical disease vectors and agriculture pragues control. Despite of its use both agriculture and human health, this bacterium can be enterotoxins producer that are also present in a few Bacillus cereus strains, emphasizing the non-haemolytic enterotoxin (NHE), haemolysin BL (HBL) and enterotoxin T (BceT) that have been related to food poisoning outbreaks reported in the literature. Thereby, this work had as a purpose to identify and to realize a genotypic characterization of these enterotoxins in one hundred B. thuringiensis strains isolated in the Amazon State, as well as to achieve the sequencing of these enterotoxin genes starting from the product of Polymerase Chain Reaction (PCR). The prevalence of these enterotoxin genes in B. thuringiensis strains by PCR method was relatively high, of which the results for the seven genes researched (bceT, hblA, hblD, hblC, nheA, nheB and nheC) showed different between themselves. By the genotypic profile were determined 27 groups and was evidenciated that 41% of the strains were positives for all the enterotoxin genes, whereas 3% were negatives for all the genes studied.. The analysis of the nucleotides and amino acids sequences of the Amazonian B. thuringiensis strains identified similarities with the nucleotides and amino acids sequences that are deposited in the GenBank and EMBL databases. / Bacillus thuringiensis é uma bactéria Gram-positiva comumente utilizada no controle de vetores de doenças tropicais e pragas da agricultura. Apesar de seu uso tanto na agricultura quanto em saúde humana, esta bactéria pode ser produtora de enterotoxinas que estão presentes também em algumas linhagens de Bacillus cereus, destacando-se a enterotoxina não-hemolítica (NHE), a hemolisina BL (HBL) e a enterotoxina T (BceT), que têm sido relacionadas a surtos de intoxicação alimentar relatados na literatura. Em vista disso, este trabalho teve como objetivo identificar e caracterizar genotipicamente estas enterotoxinas em 100 linhagens de B. thuringiensis isoladas no Estado do Amazonas, bem como realizar o seqüenciamento destes genes de enterotoxinas a partir do produto da Reação em Cadeia de Polimerase (PCR). A prevalência dos genes destas enterotoxinas nas estirpes de B. thuringiensis pelo método de PCR foi relativamente alta, cujos resultados para os sete genes pesquisados (bceT, hblA, hblD, hblC, nheA, nheB e nheC) mostraram-se distintos entre si. Pelo perfil genotípico foram determinados 27 grupos e ficou evidenciado que 41% das linhagens deram positivas para todos os genes de enterotoxinas, enquanto que 3% foram negativas para todos os genes estudados. A análise das sequências de nucleotídeos e de aminoácidos das linhagens de B. thuringiensis amazônicas identificou similaridades com as sequências de nucleotídeos e aminoácidos que estão depositadas no banco de dados do GenBank/EMBL.

Submitted by Helena Bejio (helena.bejio@unioeste.br) on 2017-11-22T12:21:24Z No. of bitstreams: 2 Dout_2016_Luciana_Iurkiv.pdf: 1597492 bytes, checksum: 9d65dcec576d4cb6c86826074cfeb38e (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-11-22T12:21:24Z (GMT). No. of bitstreams: 2 Dout_2016_Luciana_Iurkiv.pdf: 1597492 bytes, checksum: 9d65dcec576d4cb6c86826074cfeb38e (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2016-09-12 / The induced resistance efficiency in the control of pathogens is recognized, however there are costs related to its use that are still little researched, as the case of fitness and ecological costs. This research aims to verify the efficiency of use of the acibenzolar-S-methyl (ASM), mannanoligosaccharide (MOS) and B. cereus inductors in the control of spot blotch wheat disease, and its relation with the induction of enzymes related to the plant defense, as well as the interference on gas exchanges, on the non-target microrganisms A. brasilense, and on the crop production. The experiment was conduced in green house growing at the Núcleo de Estações Experimentais of the Universidade Estadual do Oeste do Paraná, Marechal Cândido Rondon Campus. The experimental design was made in randomized block design in a factorial schema 4 x 2 x 2, with four replications. The first factor "Resistance Inductors" was constituted by ASM, MOS, B. cereus inductors and water (control). The second factor "Pathogen" was constituted by the presence or absence of B. sorokiniana pathogen. The third factor "PGPR" was made up by the presence or absence of the PGPR A. brasilense. The seeds were inoculated with A. brasilense 24 h before sowing. The treatment with the resistance inductors was done 25 days after the plant emergency and 24 h after it, the inoculation with the pathogen was done. Evaluations for the quantification of peroxidase enzyme, phenylalanine ammonia-lyase and β-1.3-glucanase enzymes activities were done at 0 h, 24 h, 48 h, 72 h and 96 h after treatment. Gas exchanges were evaluated at 0 h, 24 h, 48 h, 72 h, 96 h and 120 h after treatment. The quantification of diazotrophic was made in the implementation of the experiment, at the moment of the use of the resistance inductors and in the flowering. At the flowering, biometric evaluations were performed, and at the end of the cycle, production analysis were held. The use of resistance inductors was efficient in the control of spot blotch wheat disease. The treatment with inductors did not show any interference on the endophytic diazotrophic microrganism. Higher activity of peroxidases for the MOS treatment was observed, that presented high efficiency on the control of the disease. No relation between the activities of phenylalanine ammonia lyase and β-1.3-glucanase and the control of spot blotch was observed. A higher tax of liquid assimilation of CO2 (A) in the absence of the pathogen and presence of the A. brasilense was observed, however, MOS reduced the “A”. The internal concentration of CO2 and the leaf transpiration showed as superior in the treatment with the absence of the pathogen. The stomatal conduce was affected by MOS treatment. The length of the flag leaf and leaf area were affected by ASM use. But, the total weight of grain did not suffer interference of the treatments, in spite of the weight of 100 grains to have been superior to ASM. / É reconhecida a eficiência da indução de resistência no controle de fitopatógenos, no entanto, existem custos relacionados à sua aplicação ainda pouco investigados, como é o caso dos custos adaptativos e ecológicos. No presente trabalho objetivou-se verificar a eficiência da aplicação dos indutores acibenzolar-S-metil (ASM), mananoligossacarídeo fosforilado (MOS) e Bacillus cereus no controle de mancha marrom em trigo, e sua relação com a indução de enzimas relacionadas a defesa, assim como a interferência sobre as trocas gasosas, sobre o microrganismo não alvo Azospirillum brasilense, e a produção da cultura. O experimento foi conduzido sob cultivo protegido no Núcleo de Estações Experimentais da Universidade Estadual do Oeste do Paraná, Campus de Marechal Cândido Rondon. O delineamento experimental foi em blocos ao acaso em esquema fatorial 4 x 2 x 2, com quatro repetições. O primeiro fator “Indutores de Resistência” foi constituído pelos indutores ASM, MOS, Bacillus cereus e a testemunha Água. O segundo fator “Patógeno” foi constituído pela presença ou ausência do patógeno Bipolaris sorokiniana. O terceiro fator “BPCV” constituiu-se pela presença ou ausência da bactéria promotora de crescimento vegetal A. brasilense. As sementes foram inoculadas com A. brasilense 24 h antes da semeadura. O tratamento com os indutores de resistência foi realizado 25 dias após a emergência e 24 horas após foi realizada a inoculação com o patógeno. Foram realizadas avaliações para quantificação das enzimas peroxidase, fenilalanina amônia-liase e β-1,3-glucanase às 0 h, 24 h, 48 h, 72 h e 96 horas após o tratamento. As trocas gasosas foram avaliadas às 0 h, 24 h, 48 h, 72 h, 96 h e 120 horas após o tratamento. A quantificação de diazotróficos foi realizada na implantação do experimento, no momento da aplicação dos indutores de resistência e no florescimento da cultura. No florescimento foram realizadas análises biométricas e no final do ciclo, análise dos componentes da produção. A aplicação dos indutores de resistência foi eficiente no controle da mancha marrom em trigo. Os tratamentos indutores não apresentaram interferência sobre os microrganismos diazotróficos endofíticos. Observou-se maior atividade de peroxidases para o tratamento MOS o qual apresentou grande eficiência no controle da doença. Não observou-se relação entre a atividade de fenilalanina amônia-liase e β-1,3-glucanase e o controle de mancha marrom. Observou-se maior taxa de assimilação líquida de CO2 (A) na ausência do patógeno e presença de A. brasilense, porém MOS reduziu a A. A concentração interna de CO2 e a transpiração foliar apresentaram-se superiores nos tratamentos com ausência do patógeno. A condutância estomática foi afetada pelo tratamento MOS. O comprimento da folha bandeira e a área foliar foram afetados negativamente pela aplicação de ASM. Porém, a massa total de grãos não sofreu interferência dos tratamentos, apesar de a massa de 100 grãos ter sido superior para ASM.

A análise microbiológica atua como ferramenta fundamental para a obtenção de dados sobre a qualidade, sanidade, higiene e segurança na produção de alimentos; desta forma, tem sido adotada na indústria alimentícia para o controle de qualidade. Por sua composição completa e balanceada, o leite é um substrato ideal para o desenvolvimento de diversos grupos de microrganismos. Com o objetivo de pesquisar bactérias da espécie Bacillus cereus em amostras de leite fluido, bem como a capacidade de germinação de esporos e a multiplicação dessa bactéria após processo de fervura, com manutenção das amostras à temperatura ambiente e à temperatura de refrigeração por períodos de 1, 2, 4, 6, 8, 10 e 12 horas, foram analisadas 75 amostras de leite, conforme as metodologias recomendadas por Silva et al. (2007). Destas, 46 amostras (61,3%) mostraram-se com algum grau de contaminação pela bactéria antes de serem submetidas à fervura. Por sua vez, as amostras mantidas à temperatura ambiente após a fervura, tiveram suas contagens bacterianas, principalmente a partir da 8a hora, superiores à contagem inicial, inclusive atingindo níveis capazes de desencadear uma toxinfecção alimentar, demonstrando a ocorrência da germinação dos esporos e a multiplicação das células vegetativas. Por outro lado, alíquotas dessas mesmas amostras mantidas sob refrigeração (7ºC) não atingiram populações preocupantes, enfatizando, desse modo, a importância da necessidade da refrigeração do leite após a fervura. / The microbiological analysis acts as basic tool for the attainment of data on the quality, health, hygiene and security in the food production, in such a way, she has been adopted in the nourishing industry for the quality control. For its complete and balanced composition, milk is an ideal substratum for the development of diverse groups of microorganisms. With the objective to search cereus bacteria of the Bacillus species in fluid milk samples, as well as the capacity of germination of spores and the multiplication of this bacterium after boil process, with maintenance of the samples to the ambient temperature and the temperature of refrigeration for periods of 1, 2, 4, 6, 8, 10 and 12 hours, 75 milk samples had been analyzed, as the methodologies recommended for Silva et al. (2007). Of these, 46 samples (61.3%) had revealed with some degree of contamination for the bacterium before being submitted to the boil. In turn, the samples kept to the ambient temperature after the boil, had its bacterial countings, mainly from 8a hour, superiors to the initial counting, also reaching levels capable to unchain an alimentary toxinfection, demonstrating to the occurrence of the germination of the spores and the multiplication of the vegetative cells. On the other hand, aliquot of these same samples kept under refrigeration (7ºC) had not reached preoccupying populations, emphasizing, in this manner, the importance of the necessity of the refrigeration of milk after the boil.

O Bacillus cereus é um patógeno amplamente distribuído na natureza, relacionado a intoxicações alimentares e causador da coagulação doce do leite. A intoxicação pode ocorrer após a ingestão de alimentos nos quais a bactéria produziu a toxina. Em laticínios, o B. cereus é problemático por sua habilidade de formar esporos resistentes ao calor e sobreviver aos tratamentos térmicos, como a pasteurização e UHT. As espécies de Bacillus são freqüentemente isoladas de leite pasteurizado e UHT e evidências indicam que os biofilmes formados nas superfícies dos equipamentos de processamento são uma das principais fontes de contaminação. No biofilme, as células ou esporos aderidos, têm aumentada sua resistência e podem resistir ao processo de sanificação, se tornando focos de disseminação de bactérias no processo. A matriz polimérica do biofilme reage e neutraliza os agentes sanificantes, expondo as bactérias em seu interior a doses subletais, podendo levar a aquisição de resistência. Neste estudo, biofilmes de células vegetativas B. cereus foram desenvolvidos na superfície de aço inoxidável, tipo 304, com filme de condicionamento de leite. A adesão média das células foi de 1,4 x 105UFC/cm2. Também foram desenvolvidos biofilmes de esporos, com adesão média em aço inoxidável de 1,4 x 104UFC/cm2. Foi avaliada a resistência das células vegetativas e esporos de B. cereus em biofilmes, ao processo simulado de higienização por clean in place (CIP) realizado em laticínios. Após a limpeza a adesão das células vegetativas em aço inoxidável foi reduzida a aproximadamente 1CFU/cm2. Após a sanificação, os resultados permaneceram inalterados. A contaminação remanescente foi relacionada com a maior resistência das células nos biofilmes formados. Após a limpeza e sanificação a adesão média dos esporos foi de 8,9 x 101 UFC/cm2 e 3,3 x 101 UFC/cm2, respectivamente. Tendo em vista este resultado, foi observado que esporos de B. cereus são mais difíceis de remover de superfícies de aço inoxidável do que células vegetativas, com procedimentos CIP. Finalmente, foi realizada a exposição de células vegetativas de B. cereus a três concentrações subletais (3,9; 1,8 e 0,9µg/mL) de cloreto de alquil amidopropil dimetil benzil amônio para avaliação da adaptação pelo método da concentração inibitória mínima (CIM). A aquisição de resistência foi avaliada pela comparação do valor inicial de CIM (7,4µg/mL), com os valores finais, após 28 dias de exposição. Após o período de adaptação, o valor de CIM encontrado foi o dobro do original (14,9µg/mL) indicando aquisição de resistência ao sanificante pela cepa. Foi constatado ainda que o cultivo da cepa na menor concentração subletal apresentou resultados mais coerentes, sugerindo que exposição a doses subletais mais brandas resulta em uma maior resposta adaptativa. / Bacillus cereus is a pathogen widely distributed in the environment and a serious problem in the dairy industry because of its ability to form heat-resistant spores that can survive pasteurization and UHT treatments. This bacteria is responsible for foodborne diseases in man due to production of toxins that may cause gastroenteritis, diarrhoea and vomiting. In addition, B. cereus is responsible for spoilage of pasteurized milk and cream referred to as \"sweet curdling\". Bacillus strains are frequently isolated from pasteurized and UHT treated milk and evidence indicates that biofilm formed on processing equipment surfaces are major sources of milk contamination. In biofilms, adherent cells and spores acquire increased resistance and persist to the sanitization process, becoming sources of bacteria dissemination in food processing. The extracellular polymeric matrix neutralizes the sanitizing agents and exposing the bacteria in biofilms to sublethal concentrations can lead to an increased resistance. In this study, biofilms of B. cereus vegetative cells were developed in a 304 stainless steel (SS) surface with a milk conditioning film. The mean adhesion of cells on SS surface was 1.4 x 105CFU/cm2. Biofilms of B. cereus spores were also developed, and the mean adhesion found was 1.4 x 104CFU/cm2. The resistance of B. cereus vegetative cells and spores in biofilms to a simulated clean-in-place (CIP) procedure used in dairy industry was evaluated. After cleaning procedure, the population of vegetative cells in the SS surface was reduced to approximated 1 CFU/cm2. After sanitizing procedure, the cell count remained unaltered. The remaining contamination was related with the increased resistance of cells in the developed biofilms. After cleaning and sanitizing procedures, the mean of spore adhesion found was 8.9 x 101 CFU/cm2 and 3.3 x 101 CFU/cm2, respectively. These results show that B. cereus spores are more difficult to remove from SS surfaces than vegetative cells using CIP procedures. Finally, B. cereus vegetative cells were exposed to three different sublethal concentrations (3.9; 1.8 and 0.9µg/mL) of alkyl amidopropyl dimethyl benzyl ammoniun cloride, for evaluation of adaptation, using the Minimum Inhibitory Concentration (MIC) test. The acquired resistance was evaluated by comparing the initial MIC value (7.4µg/mL) with final MICs, after 28 days of exposure. After the adaptation period, with exception of an experiment where the MIC was threefold the original value (29.8µg/mL), in the other experiments the MICs found was double (14.9µg/mL) of the original MIC, indicating acquisition of resistance by the B. cereus strain. It was observed that the growth of cells in the mildest sublethal concentration (0.9µg/mL) resulted in more consistent effects, suggesting that the exposure to mildest sublethal concentrations results in higher adaptive responses.

Bacillus cereus is a ubiquitous organism and a potential foodborne pathogen that can cause two types of gastrointestinal diseases: emesis and diarrhea. The emetic syndrome is caused by a heat and acid stable peptide toxin that is pre-formed in food, while the diarrheal syndrome is associated to two 3-protein, heat labile enterotoxin complexes that are formed in the intestine after ingestion of the organism. There are many reports on the isolation and characterization of Bacillus cereus from various foods, however there are no studies on the levels, toxigenicity and physical characteristics of B. cereus isolated from U.S. retail spices. A huge part of spices sold in the U.S. are imported from developing nations. Developing nations lack hygienic practices during processing and packaging of spices, due to which there is a high chance of imported spices being contaminated with B. cereus. Therefore, the main objective of this thesis work was to characterize B. cereus spores from U.S. retail spices. Levels of aerobic spores and B. cereus spores were determined. B. cereus spores were further analyzed for their enterotoxigenic ability, growth characteristics and physical spore characteristics. In the 247 spice samples analyzed 77 were found to contain B. cereus, while 11 were positive for B. thuringiensis. Eighty four of the 88 spices tested possessed either one of the enterotoxin genes. None of the isolates tested positive for the emetic toxin (ces) gene. Seventy five of the B. cereus isolates grew at 12 °C, although only two isolates grew well at 9 °C. Seven selected diarrheal B. cereus spore strains had D95-values ranging from 0.64-3.53 min while the two emetic strains had D95-values of 7.04 min and 6.64 min. B. cereus grew well in pre-cooked rice. After 48 h, counts of 1.26 X 107 and 3.8 X 107 B. cereus/ 10 g were obtained in pre-cooked rice maintained at 17 °C and 20 °C respectively. At 12 °C, counts did not reach 104 CFU/ 10g even after 48 h of incubation. The aerobic mesophilic bacterial population and B. cereus population of 0.1% crushed pepper in pre-cooked rice over a period of 48h at temperature 20 °C and 17 °C were also analyzed. Counts of B. cereus in pepper rice samples reached a maximum of 1600 MPN/ 10 g and 1100 MPN/ 10 g at 20 °C and 17 °C respectively while the aerobic mesophilic counts per 10 g were 2.4 X 108 and 4.4 X 106 at these temperatures. The low B. cereus counts and high aerobic mesophilic population indicates competition of nutrients in cooked rice by background flora other than B. cereus. The physical spore characteristics of five B. cereus and 3 B. thuringiensis strains were studied using transmission electron microscopy (TEM). Tubular, whip-like appendages were present in four B. cereus and two B. thuringiensis, while all seven isolates possessed exosporia.

Throughout the past decade an increased amount of attention has been drawn to the water contamination problems that affect the world. As a result, a variety of purification methods targeted at communities in developing countries have surfaced and, although all have contributed to the effort of improving water quality, few have been accepted and sustained for long term usage. Case studies indicate that the most beneficial methods are those which use indigenous resources, as they are both abundant and readily accepted by the communities. In an attempt to make a contribution to the search for water purification methods that can serve in both developed and developing countries, two fractions of mucilage gum, a Gelling (GE) and a Non-Gelling (NE) Extract, were obtained from the Opuntia ficus-indica cactus and tested as a flocculating agent against sediment and bacteria suspended in surrogate ion-rich waters. Diatonic ions are known to influence both cell binding and mucilage properties, causing CaCl2 to be tested as a flocculating agent alone and in conjunction with mucilage. Column tests were utilized to determine the settling rates of contaminant removal from the waters and the precipitated flocs were then evaluated. In columns employing Kaolin as a model for sediment removal, settling rates as high as 13.2 cm/min were observed using GE versus a control (suspensions with no treatment) settling at 0.5 cm/min. B. cereus tests displayed flocculation initiation up to 10 minutes faster than columns treated with calcium chloride (CaCl2) when using less than 10 ppm (GE) and 5 ppm (NE) of mucilage in addition to CaCl2. B. cereus removal rates between 95 and 98% have been observed in high concentration tests (> 108 cells/mL). Tests on E. coli flocculation differed slightly from those seen using B. cereus with control columns requiring 5 to 10 minutes longer to begin flocculation and mucilage treated columns displaying signs of flocculation much earlier. Mucilage is an ideal material for water purification and contaminant flocculation because it grows abundantly, is inexpensive and offers communities a sustainable technology.

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