Thesis (Ph. D.)--University of Wisconsin--Madison, 1949. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 136-145).

Techn. University, Diss., 2005--München.

Thèse (Ph.D)--Université Laval, 2003. / Titre de l'écran-titre (visionné le 22 mars 2004). Bibliogr.

Submitted by Marco Antônio de Ramos Chagas (mchagas@ufv.br) on 2017-11-06T10:14:45Z No. of bitstreams: 1 texto completo.pdf: 1239459 bytes, checksum: e1a7cd2486c370dcab988719055849df (MD5) / Made available in DSpace on 2017-11-06T10:14:45Z (GMT). No. of bitstreams: 1 texto completo.pdf: 1239459 bytes, checksum: e1a7cd2486c370dcab988719055849df (MD5) Previous issue date: 2017-06-26 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A utilização de cepas fermentadoras de Lactococcus lactis subsp. lactis produtoras de nisina em alimentos fermentados tem vantagem para a indústria pois permite um controle adicional de contaminantes, como o patógeno de origem alimentar Listeria monocytogenes. No entanto, as interações microbianas devem ser avaliadas para garantir a produção da bacteriocina no alimento e o efeito na população do patógeno. L. monocytogenes tem a capacidade de resistir a diversas condições de estresse encontradas no alimento e durante o processamento, expressando diferentes genes como o fator siga alternativo (sigB), a enzima glutamato descarboxilase (gadD), a chaperona GroEL e o transportador de glicina betaína (gbu). A exposição a uma condição de estresse em nível subletal é capaz de conferir uma maior resistência a L. monocytogenes de sobreviver a outras situações. Nesse contexto, o objetivo deste trabalho foi avaliar a expressão de genes de estresse de L. monocytogenes em interação com L. lactis subsp. lactis produtor de nisina em meio de cultura e leite. A produção de nisina em caldo BHI e leite foi avaliada por sua detecção no sobrenadante do meio de crescimento e pela expressão do gene nisK. A expressão dos genes de estresse de L. monocytogenes sigB, gadD2, groEL e gbu foi avaliada relativamente a cultura pura e na interação com L. lactis subsp. lactis. A expressão relativa dos genes de estresse de L. monocytogenes foi variável. No entanto, a expressão dos genes sigB, groEL e gbu foi inferior no tempo de 24 h durante a interação, em relação a cultura pura, o que pode indicar uma menor capacidade de sobrevivência aos estresses quando a bactéria se encontra em interação com L. lactis subsp. lactis produtor de nisina. / The use of nisin producing fermentative strains of Lactococcus lactis subsp. lactis in fermented foods has an advantage for the industry because it allows an additional control of contaminants, such as the food-borne pathogen Listeria monocytogenes. However, microbial interactions should be evaluated to ensure bacteriocin production in the food and the effect on the pathogen population. L. monocytogenes has the ability to withstand the various stress conditions encountered in food and during processing, expressing different genes such as the alternative sigma factor (sigB), the glutamate decarboxylase enzyme (gadD), the GroEL chaperone and the glycine betaine transporter (gbu). The exposure to a stress condition at the sublethal level is able to confer a greater resistance to L. monocytogenes to survive other situations. In this context, the aim of this work was to evaluate the expression of L. monocytogenes stress genes in interaction with nisin producing L. lactis subsp. lactis in culture medium and milk. The production of nisin in BHI broth and milk was evaluated by its detection in the supernatant of the growth medium and by the expression of the nisK gene. Expression of sigB, gadD2, groEL and gbu stress genes of L. monocytogenes was evaluated for pure culture and for the interaction with L. lactis subsp. lactis. The relative expression of the L. monocytogenes stress genes was variable. However, expression of the sigB, groEL and gbu genes was lower at 24 h during interaction than in pure culture, which may indicate a lower ability to survive stress when the bacterium is interacting with nisin producing L. lactis subsp. lactis.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / The objective of this study was to produce and to test the performance of native starters cultures in the elaboration of fermented Italian sausage in relation to the security and quality of the sausages. In the first experiment strains of Staphylococcus xylosus isolated from colonial sausages, were characterized and after identified for the Kit Api Staph (Biomérieux). In the second experiment, the microorganism Lactococcus lactis ssp lactis was fermented in pork plasma culture medium and evaluated its efficiency in relation to MRS broth, through microbiological analyses and optic density (OD). In the third experiment the isolated and multiplied cultures were added in dry fermented sausages, elaborating four treatments: T1 - addition of commercial starters (Staphylococcus xylosus and Lactococcus lactis ssp lactis); T2 - mixture of isolated Staphylococcus xylosus more commercial Lactococcus lactis ssp lactis ; T3 - mixture of isolated Lactococcus lactis ssp lactis more commercial Staphylococcus xylosus and T4 - Staphylococcus xylosus and Lactococcus lactis ssp lactis both isolated, evaluating its influence on the microbiological, physico-chemical and sensorial characteristics. About 13.3% of the colonies isolated of sausages were identified as Staphylococcus xylosus. The maximum growth of Lactococcus lactis ssp lactis, in pork plasma culture medium was 8.58 Log UFC.mL-1 in 27 hours, and the OD 0.88. In the MRS broth the growth reached the maximum peak (8.70 Log UFC.mL-1) in 6 hours, presenting OD of 0.81. All the elaborated sausages presented a significant decreased of pH and a reduction in the water activity, ensuring a microbiological security to the products. In relation the lipid oxidation, the treatment that contained isolated strains of Staphylococcus xylosus presented significantly lower values than the other treatments. The sausages elaborated with both strains isolated presented better sensorial results than the sausages elaborated with commercial starters cultures. Therefore, this study indicated that would be promising to extend the availability of microorganisms for industrial use from the selection of native cultures, that the pork plasma culture medium becomes an alternative for the multiplication of lactic acid bacteria, because it presented a similar fermentation performance to the commercial culture medium and that the addition of natives starters cultures can be used in the elaboration of fermented Italian sausages, providing safety products and with differentiated flavor. / Este trabalho teve por objetivo produzir e testar o desempenho de culturas starters nativas na fabricação de salame tipo Italiano quanto à segurança e qualidade dos salames. No primeiro experimento, cepas de Staphylococcus xylosus foram isoladas de salames coloniais, caracterizadas e após identificadas pelo Kit Api Staph (Biomérieux). No segundo experimento, o microrganismo Lactococcus lactis ssp lactis foi fermentado em meio de cultura de plasma suíno e avaliado sua eficiência em relação ao caldo MRS, através de análises microbiológicas e densidade óptica (DO). No terceiro experimento, as culturas isoladas e multiplicadas foram adicionadas em salame, elaborando-se quatro tratamentos: T1 - adição de starters comerciais (Staphylococcus xylosus e Lactococcus lactis ssp lactis); T2 - mistura de Staphylococcus xylosus isolado mais Lactococcus lactis ssp lactis comercial; T3 - mistura de Lactococcus lactis ssp lactis isolado mais Staphylococcus xylosus comercial e T4 - Staphylococcus xylosus e Lactococcus lactis ssp lactis ambos isolados, avaliando sua influência nas características microbiológicas, físico-químicas e sensoriais. Foram identificadas 13,3% das colônias isoladas de salames coloniais como Staphylococcus xylosus. O crescimento máximo de Lactococcus lactis ssp lactis, em meio de cultura de plasma suíno, foi de 8,58 Log UFC.mL-1 no tempo de 27 horas e DO de 0,88. Em caldo MRS, o crescimento atingiu pico máximo (8,70 Log UFC.mL-1) em 6 horas, apresentando uma DO de 0,81. Todos os salames elaborados apresentaram uma queda de pH significativa e também uma redução na atividade de água, garantindo uma segurança microbiológica aos produtos. Em relação a oxidação lipídica, os tratamentos que continham cepas de Staphylococcus xylosus isolados apresentaram valores significativamente menores que os outros tratamentos. Os salames elaborados com as duas cepas isoladas (T4) apresentaram melhores resultados sensoriais quando comparados com salames elaborados com culturas starters comerciais. Portanto, este estudo indicou que seria promissor ampliar a disponibilidade de linhagens para uso industrial proveniente da seleção de culturas nativas, que o meio de cultura de plasma suíno torna-se uma alternativa para a multiplicação de bactérias ácido lácticas, pois apresentou um desempenho semelhante à fermentação do meio de cultura comercial e que a adição de culturas starters nativas pode ser utilizada na elaboração de salames, proporcionando produtos seguros e com flavor diferenciado.

Résumé confidentiel / Résumé confidentiel

Submitted by Reginaldo Soares de Freitas (reginaldo.freitas@ufv.br) on 2015-11-09T08:55:27Z No. of bitstreams: 1 texto completo.pdf: 1029951 bytes, checksum: 361758ed3d2e0d1b698e70ceef444093 (MD5) / Made available in DSpace on 2015-11-09T08:55:27Z (GMT). No. of bitstreams: 1 texto completo.pdf: 1029951 bytes, checksum: 361758ed3d2e0d1b698e70ceef444093 (MD5) Previous issue date: 2007-08-06 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / O peptídeo denominado SBm 7462 (Sintético Boophilus microplus 7462) é uma vacina desenvolvida a partir da seqüência da proteína intestinal do carrapato B. microplus denominada Bm 86 apresentando ação imunogênica contra o mesmo. O carrapato B. microplus é um importante artrópode da medicina veterinária devido a perdas econômicas e problemas de saúde causados pelo referido parasita no gado da América Central e do Sul, bem como da Austrália. Este trabalho descreve a obtenção de linhagens de Kluyveromyces lactis recombinantes produtoras do referido peptídeo. Dois vetores de expressão integrativos foram construídos a partir do vetor pKLAC1: pKLAC1-seq1, contendo a seqüência 1 constituída por três cópias da seqüência de nucleotídeos que codifica para o SBm 7462 e o pKLAC1-seq4, contendo a seqüência 4 constituída por uma única cópia da seqüência de nucleotídeos que codifica o SBm 7462. As construções foram confirmadas por sequenciamento. O cassete de integração obtido pela clivagem do vetor com a enzima SacII foi utilizado na transformação da linhagem selvagem de K. lactis CBS2359. A integração dos cassetes por recombinação homóloga no locus LAC4 foi confirmada por PCR. A análise dos recombinantes de K. lactis por RT-PCR e a detecção do peptídeo, utilizando anticorpos monoclonais contra SBm 7462, comprovaram a transcrição e a expressão extracelular dos peptídeos. Os recombinantes foram cultivados em regime de batelada em YPGlicerol, alcançando uma concentração de biomassa de 10 g·L-1. A biomassa obtida foi transferida para o meio YNB com 4% (p/v) de lactose para induzir o promotor LAC4 e a síntese de SBm 7462 recombinante. Amostras foram analisadas quanto à concentração de proteína extracelular, por eletroforese em SDS-PAGE e detecção do imunógeno. A indução da síntese dos peptídeos foi testada sob duas condições: sem pulso adicional de lactose e com pulso de lactose, a cada 24 horas, durante 144 horas de indução. A concentração de proteínas extracelulares na segunda condição foi maior quando comparada com a condição sem pulso adicional de lactose. Atividade proteolítica não foi detectada no sobrenadante. O perfil eletroforético em SDS-PAGE revelando três bandas de aproximadamente 6, 10 e 21 kDa relacionadas ao peptídeo de interesse é discutido. A detecção do peptídeo vacinal com anticorpos monoclonais comprovou a presença extracelular do rSBm 7462. / The peptide designated SBm 7462 (Synthetic Boophilus microplus 7462) induces an immunogenic response against the tick B. microplus, an important arthropods in veterinary medicine due to the economic losses and the health problems caused in cattle production in Central and South America and Australia. Here we describe the construction of recombinant Kluyveromyces lactis yeast strains expressing the gene for rSBm 7462. Two integrative expression cassettes harboring one or three copies of the nucleotide sequence coding for the SBm 7462 were built from the vector pKLAC1. The construction of the expression vectors was confirmed by sequencing. The transformation of wild type K. lactis CBS2359, was performed and the integration of the cassettes by homologous recombination into the LAC4 locus of the transformed K. lactis genomes was verified by PCR. The transcription and extracellular expression of the peptides have been confirmed in recombinants K. lactis strains by RT-PCR and monoclonal antibodies against SBm 7462. The recombinants were cultured in shake-flask YPGlycerol medium generating 10 g·L-1 biomass. The biomass obtained was transferred to mineral medium with lactose 4% (w/v) to induce the LAC4 promoter and the synthesis of recombinant SBm 7462. Samples were analyzed for total protein, SDS-PAGE and immunogenic detection. The peptide production was investigated under two conditions: with additional lactose pulse at 24 hours intervals during 144 hours of induction and with no additional lactose pulse. The extracellular protein concentration in the first condition was higher compared to the condition with no additional pulse of lactose. Proteolytic activity was not detected into the medium. Three major protein bands of approximated 6, 10 and 21 kDa related to the desired peptide were detected by SDS-PAGE.

Diploma thesis was focused on identification of lactic acid bacteria of species Lactococcus lactis and subspecies Lactococcus lactis ssp. lactis and Lactococcus lactis ssp. cremoris using species and subspecies specific polymerace chain reaction (PCR). PCR method was used for identification of bacteria of species Lactococcus lactis in 10 samples of hard cheeses. The method of sample preparation was evaluated for hard cheeses with the aim to receive sufficient amount of cells for the preparation of crude cell lysates. Whole DNA in quality suitable for PCR was separated using magnetic microspheres P(HEMA-co-GMA) in the presence of polyethylenglycol (PEG 6000) and sodium chloride. DNA isolated by phenol extraction was used as control of DNA isolation. PCR was used to the analysis of 7 strains of Lactococcus lactis from Collection of dairy microorganisms Laktofora (CCDM). Altogether 5 or 2 strains were identified into subspecies Lactococcus lactis ssp. lactis and Lactococcus lactis ssp. cremoris, respectively.

The proteolytic system of Lactococcus lactis starter cultures influences both flavor and the characteristic body and texture of cheese. The ability to further understand and control how different components of this proteolytic system work together to hydrolyze milk proteins would be of immense importance to the dairy industry. The goal of this research was to characterize Lactococcus lactis subsp. lactis starter bacteria with varying prt operon compositions by proteinase specificity, aminopeptidase and lipase activities, growth, and influence on cheese flavor. By using a cheese slurry system, a statistical model to predict milk protein hydrolysis patterns was developed. Lactococcus lactis subsp. lactis C20 has five plasmids of 55 (pJK550), 48 (pJK480), 43 (pJK430), 3.7 (pJK037), and 2.1 (pJK021) kilo bases. Two of these plasmids (pJK550 and pJK430) are necessary for full proteolytic capability, i.e., clotting milk in 16 h at 20°C. Plasmid pJK550 codes for a proteinase that catalyses the first step in casein degradation. Plasmid pJK430 codes for an oligopeptide transport system, which further transports peptides across the membrane for bacterial metabolism. Strains were constructed containing twelve different combinations of proteolytic phenotypes, such as Lac+PrtP+Opp+, Lac+PrtP+Opp-, Lac+PrtP-Opp+, Lac+Prt-Opp-, Lac-PrtP+Opp+, Lac-PrtP+Opp-, Lac-Prt-Opp+, and Lac-Prt-Opp-. The proteinase specificities of these strains toward milk proteins were dependent on the genotypes present. Genetically all strains showed a P1-type proteinase. Enzymatically C20 had group g proteinase specificity, whereas the rest of the strains containing the proteinase gene showed mixed group specificity. a a-Casein was only slightly hydrolyzed by all strains. B-Casein had a variable pattern, as did mixed casein and milk. K-Casein hydrolysis showed similar degradation patterns in all strains except CB06, which varied in its profile from the other strains. Sensory evaluation showed that culture had a significant effect on rancidity but not on acidity or bitterness. It also showed that the proteolytic system was associated with lipase activity in these strains. A statistical prediction model was developed that allowed strains to be classified according to their amino acid hydrolysis patterns. Mixed casein solution proved to be the best substrate for this analysis. Relationships among strains were seen more easily with canonical analysis and distance tables than by looking only at amino acid hydrolysis patterns.

Chapter I reviews current literature on gene transfer systems in lactic acid bacteria, how genetically altered microorganisms for food are presently regulated, and how nisin is used as a food preservative. Chapter II investigates previous reports which linked genes for nisin biosynthesis and sucrose utilization (Nip+Suc+) to plasmid DNA in two well characterized L· lactis subsp. lactis strains. Plasmid curing studies, conjugations, and DNA-DNA hybridizations indicated that these genes were encoded by chromosomal loci in all Nip+Suc+ strains examined. Similar results were noted in nisin-sucrose transconjugants of L. lactis subsp. cremoris and S. salivarius subsp. thermophilus in Chapters III and IV. Chapter III describes the use of conjugation to construct nisin-producing Lactococcus lactis subsp. cremoris strains. The direct-plate conjugation method was developed to facilitate transfer of Nip+Suc+ to L. lactis subsp. cremoris recipients. DNA-DNA hybridizations to transconjugant DNAs with an oligonucleotide that detected the nisin structural gene, nisA, demonstrated that this gene was transferred during conjugation. Lactococcus lactis subsp. cremoris Nip+Suc+ transconjugants retained the recipient strain phenotype with respect to bacteriophage resistance and acid production in milk. These results indicated that it would be feasible to construct nisin-producing L. lactis subsp. cremoris strains for mixed and multiple starter systems. Chapter IV investigates features of Nip+Suc+ transfer using a Lactococcus lactis subsp. lactis model system. Intergeneric transfer of nisin-sucrose genes was also achieved between lactococcal Nip+Suc+ donors and Streptococcus salivarius subsp. thermophilus recipients. Streptococcal transconjugants acquired Suc+ and nisin immunity but did not produce nisin. DNA-DNA hybridizations, however, demonstrated that nisA was present in these transconjugants. To investigate whether nisA was involved in nisin immunity, this gene was cloned and electro-transformed into Lactococcus lactis subsp. lactis LM0230. Electro-transformants did not express nisin immunity or any other trait linked to nisin production in lactococci. Results presented in Chapter V indicate that nisin may have application for control or prevention of bovine mastitis. Gram-positive pathogens which cause bovine mastitis were examined for their susceptibility to nisin. Disc diffusion assays indicated that minimum inhibitory concentrations of nisin ranged from 10 to 250 ug per ml. In addition, 50 ug of nisin per ml in milk inhibited all gram-positive pathogens tested.