Degradation of peroral administered drugs by various enzymes in the gastrointestinal tract has proven to be troublesome for the absorption' and bioavailability of protein and peptide drugs. Mucoadhesive polymers such as poly(acrylates) have proven to inhibit protease enzymes responsible for initiating digestion of peptide drugs. Enzyme inhibitors have unique chemical properties enabling it to interact with enzymes to form complexes with such enzymes prohibiting it from functioning properly. Anionic carboxymethylated chitosan derivatives such as N,N-dicarboxymethyl chitosan and N, O-carboxymethyl chitosan display unique structural similarities to enzyme inhibitors being anionic polymers that may interact with bi-valent cations... / Thesis (M.Sc. (Pharm.))--North-West University, Potchefstroom Campus, 2004.

The mechanisms by which altered processing, distribution and secretion of proteolytic enzymes occur, facilitating degradation of the extracellular matrix in invasive and metastatic cells, are not fully understood. Studies on the MCF-10 A breast epithelial cell line and its premalignant, c-Ha-ras-transfected MCF-10AneoT counterpart have shown that the ras-transfected cell line has a more alkaline pH. The objective of this study was to determine which organelles of the endosome-lysosome route were alkalinized and shifted to the cell periphery after ras-transfection. Antibodies to the hapten 2,4-dinitrophenyl (DNP), required for pH studies, were raised in rabbits and chickens using DNP-ovalbumin (DNP-OVA) as immunogen. Cationised DNP-OVA (DNP-catOVA) was also inoculated to increase antibody titres. Anti-hapten and carrier antibody titres were assessed. In rabbits, cationisation seems useful to increase anti-DNP titres if a non-self carrier protein (OVA) is used. In chickens, cationisation of DNP-OVA seems necessary to produce a sustained anti-OVA (anti-self) response (implying a potential strategy for cancer immunotherapy). Oregon Green® 488 dextran pulse-chase uptake and fluorescent microscopy, and (2,4-dinitroanilino)-3'-amino-N-methyldipropylamine (DAMP) uptake, immunolabelling for DNP (a component of DAMP) and unique markers for the early endosome (early endosome antigen-I, EEAI), the late endosome (cation-independent mannose-6-phosphate receptor, CI-MPR) and the lysosome (small electron dense morphology and lysosome-associated membrane protein-2, LAMP-2) and electron mlcroscopy was performed. The pH of late endosomes and lysosomes in the ras-transfected MCF-10AneoT cell line were found to be relatively alkalinised and Iysosomes shifted toward the cell periphery. The acidic pH of late endosomes is required to release precursor cysteine and aspartic proteases from their receptors (e.g. CI-MPR), process the precursors to active proteases and to allow receptor recycling. The more alkaline pH observed potentially explains the altered processing of proteases in rastransfected cells. Alkalinisation ofthe cytosol may affect the cytoskeleton responsible for, among other things, the positioning and trafficking of various organelles, causing relocation of Iysosomes toward the cell periphery and actin depolymerisation. This may enable fusion of Iysosomes with the plasma membrane and the release of proteolytic enzymes, facilitating the observed invasive phenotype. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2005.

Infectious bursal disease virus (IBDV) is a birnavirus that infects the B-cells in the bursa of Fabricius of young chickens, causing Gumboro disease. The IBDV 114 kDa polyprotein (NH2-pVP2-VP4-VP3-COOH) is thought to be processed at 512Ala-Ala513 and 755Ala-Ala756 through the proteolytic activity of VP4, a serine protease which uses a Ser/Lys catalytic dyad, to release pVP2, VP4 and VP3. Precursor VP2 (pVP2) is further processed at its C-terminus to generate VP2 and structural peptides through the cleavage of the 441Ala-Phe442, 487Ala-Ala488, 494Ala-Ala495 and 501Ala-Ala502 peptide bonds to release VP2 and four structural peptides, pep46, pep7a, pep7b and pep11. While the processing at the 441Ala-Phe442 site was shown to be mediated by the endopeptidase activity of VP2, the processing at the other two sites is not well understood. The products resulting from the processing of the IBDV polyprotein were previously identified by anti-VP2 and anti-VP3 antibodies. The present study used anti-VP4 peptide antibodies to identify products resulting from the IBDV polyprotein processing. It was hypothesised that VP4 exists in two forms, the embedded form which exists as an integral part of the polyprotein and a mature form which is released after the processing. In order to characterise the two forms of VP4, six different fragments i.e. full-length polyprotein (Met1-Glu1012), truncated polyprotein (Ile227-Trp891), VP4-RA (Arg453-Ala755), VP4-RK (Arg453-Lys722), VP4-ΔVP3 (Ala513-Trp891, called VP4-AW for the sake of simplicity) and VP4-AA (Ala513-Ala755) were amplified from the IBDV dsRNA, cloned into a T-vector and sub-cloned into several expression vectors. The constructs were sequenced prior to expression. The sequence of the polyprotein coding region was used to determine the pathotype of the isolate used for viral dsRNA isolation. This isolate was from IBDV-infected bursae harvested from commercial chickens during an IBD outbreak in KwaZulu-Natal, South Africa in 1995, thus naming the isolate SA-KZN95. The comparison of the deduced amino acid sequence of SA-KZN95 polyprotein with 52 sequences of other IBDV strains highlighted 21 residues which could be molecular markers of different IBDV pathotypes. The residues of SA-KZN95 were identical to those of the Malaysian very virulent UPM94/273 strain. The constructs representing the embedded and mature forms of VP4 were recombinantly expressed. Processing was observed from the expression of the full-length polyprotein, truncated polyprotein, VP4-RA, VP4-RK and VP4-AW, but not from VP4-AA expression. The mutation of the Ser/Lys catalytic dyad in the full-length polyprotein, truncated polyprotein, VP4-RA, VP4-RK and VP4-AW, prevented processing thus verifying that the proteolytic activity was due to VP4. Anti-VP4 peptide antibodies were raised in chickens for the identification of the polyprotein cleavage products. The anti-VP4 peptide antibodies detected more cleavage products than expected from the polyprotein, suggesting that additional or different cleavage sites may be used. The characterisation of the cleavage products suggested that the processing for the release of VP4 occurs either at the 487Ala-Ala488 or the 512Ala-Ala513 site in a single polyprotein molecule. Ultimately, an IBDV polyprotein processing strategy that would explain the release of the additional products was proposed in the present study. The present study also illustrated the importance of Pro377 in the processing of the polyprotein where its replacement with Leu induced a prominent change in polyprotein processing. The mutation seemed to induce structural changes that may possibly affect the cleavage sites. Although no autocatalytic activity was observed during the expression of VP4-AA (mature form), it cleaved mutant VP4-RK in trans. It seemed to be active as a dimer on a gelatine gel but no activity was observed against a dialanyl fluorogenic peptide substrate. It also appeared to form peptidase-inhibitor complexes with anti-thrombin III. The present study also describes attempts to detect native VP4 in IBDV-infected bursa homogenates by anti-VP4 peptide antibodies on a western blot and by proteolytic activity determination on gelatine-containing SDS-PAGE gels. The findings of the study provide new information that may contribute to the development of anti-viral agents. These anti-viral agents may target polyprotein processing, capsid assembly and thus prevent virus replication during IBDV infection. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2011.

Degradation of peroral administered drugs by various enzymes in the gastrointestinal tract has proven to be troublesome for the absorption' and bioavailability of protein and peptide drugs. Mucoadhesive polymers such as poly(acrylates) have proven to inhibit protease enzymes responsible for initiating digestion of peptide drugs. Enzyme inhibitors have unique chemical properties enabling it to interact with enzymes to form complexes with such enzymes prohibiting it from functioning properly. Anionic carboxymethylated chitosan derivatives such as N,N-dicarboxymethyl chitosan and N, O-carboxymethyl chitosan display unique structural similarities to enzyme inhibitors being anionic polymers that may interact with bi-valent cations... / Thesis (M.Sc. (Pharm.))--North-West University, Potchefstroom Campus, 2004.

Thesis (Ph.D. (Chemistry)) - Rhodes University, 2008.

Made available in DSpace on 2014-06-11T19:22:54Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-02-23Bitstream added on 2014-06-13T18:49:55Z : No. of bitstreams: 1 zanphorlin_lm_me_sjrp.pdf: 945446 bytes, checksum: 9959e6f0472c3ac5cdce9a3374c74387 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fungos termofílicos têm despertado grande interesse acadêmico e industrial por produzirem uma variedade de enzimas termoestáveis com potenciais aplicações em processos biotecnológicos como biocatálise nas indústrias de couro, farmacêutica, têxtil e alimentícia, e na preparação de produtos de limpeza e cosméticos. Particularmente, as proteases, além de participarem de inúmeros processos fisiológicos vitais como vias metabólicas, hemostasia e sinalização celular, também representam hoje cerca de 60% do mercado mundial de enzimas. Neste trabalho, descrevemos a produção, purificação e caracterização bioquímica de uma serino-protease produzida por um fungo termofílico do gênero Myceliophthora. As taxas de atividade proteolítica foram avaliadas através de fermentação em meio sólido (FES) e submerso (FSM) e observou-se um rendimento na atividade proteolítica 4,5 vezes maior para o meio sólido. A enzima bruta obtida por ambos os procedimentos (FES e FSM) exibiu a mesma temperatura ótima de 50 ºC, porém em relação ao pH ótimo houve um deslocamento de 7 (FSM) para 9 (FES) sugerindo que o perfil enzimático do fungo difere de acordo com suas condições de fermentação. Baseado nesses resultados prosseguiu-se os estudos com o extrato bruto obtido por FES. A imobilização da enzima bruta em esferas de alginato de cálcio resultou no aumento da temperatura ótima e na estabilidade térmica quando comparado com a enzima livre. O extrato bruto obtido por FES foi, então, fracionado por métodos cromatográficos como exclusão molecular e troca-iônica que resultaram na protease pura com peso molecular de 28,2 kDa determinado por espectrometria de massa. A protease pura demonstrou pH ótimo de 9,0 e temperatura ótima de 45 °C que corroboram... / Thermophilic fungi have attracted great academic and industrial interest because they produce a variety of thermostable enzymes with potential applications in biotechnological processes such as biocatalysis in the industries of leather, pharmaceutical, textile and food, and the preparation of detergents and cosmetics. In particular, proteases not only participate in many vital physiological processes such as metabolic pathways, cell signaling and homeostasis, but also currently represent about 60% of the world market of enzymes. In this work, we describe the production, purification and biochemical characterization of a serine protease produced by a thermophilic fungus of the genus Myceliophthora. The levels of proteolytic activity were evaluated either by solid fermentation (SSF) and submerged (SmF). The crude enzyme obtained by both procedures (SSF and SmF) exhibited similar optimum temperature of around 50 ºC, but in relation to the optimum pH was shifted of 7 (SmF) to 9 (SSF), suggesting that the enzymatic profile of the fungus differs from according to its fermentation conditions. Based on these results, the studies were followed with crude extract obtained by SSF. The immobilized enzyme on beads of calcium alginate resulted in increased optimum temperature and thermal stability when compared to the free enzyme. The crude extract obtained by SSF was then fractionated by chromatographic methods including molecular exclusion and ion-exchange that resulted in the pure protease with molecular weight of 28.2 kDa as determined by mass spectrometry. The pure protease showed optimum pH of 9.0 and optimum temperature of 45 °C that corroborate to the preliminary characterization of the crude extract. Inhibition tests resulted in complete inhibition by PMSF, a canonical... (Complete abstract click electronic access below)

Submitted by Vasti Diniz (vastijpa@hotmail.com) on 2017-09-06T13:04:06Z No. of bitstreams: 1 arquivototal.pdf: 1188137 bytes, checksum: 010a2962d49aa738285e5f27b77c15b3 (MD5) / Made available in DSpace on 2017-09-06T13:04:06Z (GMT). No. of bitstreams: 1 arquivototal.pdf: 1188137 bytes, checksum: 010a2962d49aa738285e5f27b77c15b3 (MD5) Previous issue date: 2015-08-31 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The microbial communities play a fundamental role in the health of coral and their changes can lead to the onset of disease. Many diseases affect coral reefs in several parts of the world, however, little is known about the pathogenic agents and the factors that trigger the pathological process. In coastal reefs of Carapibus Beach of Conde (Paraiba state, Brazil), a tissue necrosis affects the zoanthid Palythoa caribaeorum. This study aimed to compare the diversity of culturable bacteria in the samples of healthy and necrotic tissue from the P. caribaeorum colony collected from the Carapibus reefs. The present work aimed also to analyze the production of proteolytic enzymes by isolated bacteria. The density of total heterotrophic bacteria in the necrotic tissue was higher than in healthy tissue of P. caribaeorum. Phylogenetic analysis of isolated bacteria based on the partial 16S rRNA gene sequences revealed that the majority of bacterial strains belonged to the Bacilli class of Firmicutes phylum (65.2%), followed by the Gamma Proteobacteria class (34.7%) of Proteobacteria phylum. The genus Bacillus was dominant among the strains of healthy tissue of P. caribaeroum, while among the bacteria of necrotic tissue the Bacillus and Vibrio were the most abundant. The higher number of strains belonged to the Vibrio genus were found in necrotic tissue (42.1%) in comparison with healthy tissue (9.6%). Among bacteria from healthy tissue were found also Staphylococcus sp. isolate and from necrotic tissue Marinobacter spp. and Alteromonas spp. isolates. Among the isolates from healthy tissue only Bacillus spp. showed proteolytic activity, while proteolytic isolates from necrotic tissue belonged to the genera: Bacillus, Vibrio, Alteromonas and Marinobacter. The data suggest that bacteria of the genus Vibrio and proteolytic bacteria may play a role in the development of tissue necrosis of zoanthid studied. / As comunidades microbianas desempenham um papel fundamental na saúde do coral, e suas alterações podem levar ao aparecimento de doenças. Muitas doenças afetam os corais dos recifes em várias partes do mundo, entretanto pouco se sabe sobre os agentes patogênicos e os fatores que desencadeiam o processo patológico. Nos recifes costeiros da praia de Carapibus, Conde (Paraiba, Brasil), uma doença necrosante afeta o zoantideo Palythoa caribaeorum. Em virtude disso, neste trabalho objetivou-se comparar a diversidade de bactérias cultiváveis nas amostras do tecido sadio e necrosado coletadas da colônia de P. caribaeorum dos recifes de Carapibus. Objetivou-se também analisar a produção de enzimas proteolíticas por bactérias isoladas. A densidade de bactérias heterotróficas totais no tecido necrosado foi maior que no tecido sadio. A análise filogenética dos isolados bacterianos realizada na base das sequencias parciais do gene RNAr 16S revelou que o maior número de isolados de bactérias pertenceram a classe Bacilli do filo Firmicutes (65,2%), seguida pela classe Gama-Proteobacteria (34,7%) do filo Proteobacteria. O gênero Bacillus foi predominante entre os isolados do tecido sadio de P. caribaeroum, enquanto entre as bactérias do tecido necrosado os gêneros Bacillus e Vibrio foram dominantes. Um número maior de isolados pertencentes ao gênero Vibrio foi encontrado no tecido necrosado (42,1%) em relação ao tecido sadio (9,6%) de P. caribaeorum. Foram encontradas também as bactérias Staphylococcus sp. no tecido sadio, Marinobacter spp. e Alteromonas spp. no tecido necrosado. Dentre os isolados do tecido sadio de P. caribaeorum, apenas o gênero Bacillus apresentou a atividade proteolítica, enquanto os isolados proteolíticos do tecido necrosado pertenceram aos gêneros: Bacillus, Vibrio, Alteromonas e Marinobacter. Os dados obtidos sugerem que as bactérias do gênero Vibrio e as bactérias proteolíticas podem desempenhar um papel no desenvolvimento da doença necrosante do zoantídeo estudado.

A produção de bacteriocina por Bactérias Ácido Lácticas tem atraído grande atenção por causa do seu status GRAS (Generally Recognized as Safe), e seu uso potencial como aditivo seguro para a conservação de alimentos. Em função de suas características antimicrobianas, as bacteriocinas têm sido testadas como bioconservadores em diversos produtos, mostrando atividade contra microrganismos patogênicos. Porém, o elevado custo de produção ainda tem sido um fator relevante para ampla utilização deste tipo de conservante. Portanto, o objetivo deste trabalho foi avaliar a viabilidade da produção de enterocinas, utilizando soro de leite parcialmente desmineralizado e água de maceração de milho (milhocina) como substrato. Foram avaliados 5 isolados de enterococos (Efm20, Efm22, Efm24, Efm25 e Efs27) produtores de enterocina em meio MRS frente a bactéria Listeria innocua CLIP 12612 e Listeria monocytogenes 2032. O meio de cutura MRS foi substituido na concentração de 25% por soro de leite ou milhocina. O ensaio da ação da enterocina foi realizado pelo método de difusão em poços. O caráter proteico da bacteriocina foi confirmado após incubação com enzimas proteolíticas α-quimiotripsina, protease e proteinase-K. A produção de peróxido de hidrogênio e ácido lático foram descartadas pela utilização de catalase e neutralização com NaOH. O sobrenadante livre de células (CFS) obtido em ambos substratos foram tratados a 80 °C e 100 °C, apresentando termoestabilidade. Os CFS obtidos em MRS com milhocina pelo isolado Efm22 (24 horas) atingiu 6400 UA/mL frente às duas bactérias indicadoras. Já os isolados Efm24 (24 horas) e Efm20 (18 horas) chegaram a 6400 UA/mL frente a Listeria innocua CLIP 12612. Em soro de leite, os maiores valores de UA/mL ocorreram pelos isolados Efm20, Efm25 e Efs27 (24 horas). Em conclusão, constatou-se que os substratos testados apresentam potencial para serem aplicados na fabricação de bioconservantes de produtos alimentícios. / The production of bacteriocin by Lactic Acid Bacteria has attracted great attention because of its GRAS (Generally Recognized as Safe) status, and its potential use as a safe additive for food preservation. Due to their antimicrobial characteristics, bacteriocins have been tested as bioconservatives in several products, showing activity against pathogenic microorganisms. However, the high cost of production has still been a relevant factor for the wide use of this type of preservative. Therefore, the objective of this work was to evaluate the viability of enterocin production using partially demineralized whey and corn steep liquor as substrate. Five Enterococcus isolates (Efm20, Efm22, Efm24, Efm25 and Efs27) were tested on MRS medium against Listeria innocua CLIP 12612 bacterium and Listeria monocytogenes 2032. The MRS culture medium was replaced at 25% concentration by whey or corn steep liquor.The assay of the action of enterocin was performed by the well diffusion method. The proteinic character of bacteriocina was confirmed after incubation with proteolytic enzymes α-chymotrypsin, protease and proteinase-K. The production of hydrogen peroxide and lactic acid were discarded by the use of catalase and neutralization with NaOH. The cell free supernatant (CFS) obtained on both substrates were treated at 80 ° C and 100 ° C, exhibiting thermostability. The CFS obtained in MRS with corn steep liquor by the Efm22 isolate (24 hours) reached 6400 AU / mL against the two indicator bacteria. On the other hand, the isolates Efm24 (24 hours) and Efm20 (18 hours) reached 6400 AU / mL compared to Listeria innocua CLIP 12612. In whey, the highest values of UA / mL occurred with the isolates Efm20, Efm25 and Efs27 (24 hours). In conclusion, it was verified that the substrates tested have potential to be applied in the manufacture of food product bioconservants.

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / A produÃÃo de enzimas por fermentaÃÃo semi-sÃlida (FSS) à influenciada por diversos fatores de cultivo que afetam o crescimento microbiano e a produÃÃo de metabÃlitos. O estudo de fatores como aeraÃÃo e umidade do ar torna-se indispensÃvel para a otimizaÃÃo deste bioprocesso. Neste contexto, o presente trabalho teve como objetivo avaliar a produÃÃo de protease pelo fungo Aspergillus oryzae CCBP001 por FSS em biorreator de colunas. A FSS para a produÃÃo de protease utilizando o fungo filamentoso A. oryzae CCBP 001 ocorreu em condiÃÃes dinÃmica e estÃtica, visando observar o mÃtodo que apresentou a maior produÃÃo. Para tanto foram testados os resÃduos agroindustriais: torta de canola, torta de girassol, farelo de trigo, pelÃcula da amÃndoa de caju e farelo de algodÃo como substrato, observando o perfil de produÃÃo em funÃÃo de diferentes atividades de Ãgua (Aw) iniciais obtidas pela adiÃÃo de distintos volumes de Ãgua para umidificaÃÃo. A produÃÃo de protease em reator de colunas nas condiÃÃes otimizadas foi comparada com a produÃÃo em Erlenmeyer, durante dez dias. Foram avaliados procedimentos para recuperar, identificar, concentrar, e estocar o extrato enzimÃtico produzido. Para a concentraÃÃo do extrato enzimÃtico produzido realizou-se estudo de secagem em âspray dryerâ e acompanhou-se o tempo de estocagem do extrato seco durante 90 dias. Com os resultados foi possÃvel selecionar a torta de canola como o substrato onde apresentou uma produÃÃo 33% superior aos demais substratos testados. No estudo das condiÃÃes operacionais em reator de colunas foi possÃvel avaliar a influÃncia da vazÃo do ar, umidade relativa do ar e umidade do substrato na produÃÃo de protease. A utilizaÃÃo de glicose, maltodextrina e carboximetilcelulose como adjuvantes se mostraram eficientes com relaÃÃo à manutenÃÃo da atividade de protease durante o processo de secagem utilizando âspray dryerâ, onde foi possÃvel obter um produto seco com baixos valores de umidade e Aw, importante para o processo de estocagem do extrato enzimÃtico. A secagem por atomizaÃÃo do extrato enzimÃtico possibilitou concentrar e estocar a enzima. / Enzyme production by solid state fermentation (SSF) is influenced by several factors that affect crop growth and production of microbial metabolites. The study of factors such as aeration and moisture in the air becomes indispensable for this bioprocess optimization. In this context, the present study aimed to assess the protease production by Aspergillus oryzae CCBP001 by FSS bioreactor columns. The FSS for the production of protease using the filamentous fungus A. oryzae CCBP 001 occurred in dynamic and static conditions in order to observe the method with the highest production. Therefore, tested the agroindustrial waste: canola cake, sunflower cake, wheat bran, almond cashew film and cottonseed meal as substrate, observing the production profile for different water activity (Aw) obtained by initial adding different volumes of water for humidification. Protease production in reactor columns in the optimized conditions was compared to production in flasks for 10 days. Evaluated procedures to recover, identify, focus and store the enzyme extract produced. For the concentration of the enzyme extract produced a study was conducted in dry "spray dryer" and followed up the storage time of the dry extract for 90 days. From the results it was possible to select the canola cake as the substrate where it presented a production 33% higher than the other substrates tested. In the study of operating conditions in reactor columns was possible to evaluate the influence of air flow, air humidity and substrate moisture in protease production. The use of glucose, maltodextrin and carboxymethylcellulose as adjuvants proved to be efficient with regard to maintenance of protease activity during the drying process using a "spray dryer", where it was possible to obtain a dry product with low values of humidity and Aw important for the storage process of enzyme extract. Spray drying of the enzyme extract and concentrate stockpile allowed the enzyme.

Made available in DSpace on 2017-07-10T17:48:05Z (GMT). No. of bitstreams: 1 Elisangela_Borsoi_Pereira.pdf: 4099678 bytes, checksum: 2154628e3fc69ca40c985e332b48f374 (MD5) Previous issue date: 2014-09-26 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Two experiments were conducte, in order to identify the best time for production and maturation of Traditional Colonial and Imbriago Colonial cheese in the municipality of Pinhão - Paraná. Ten Holstein heifers, grazing on pasture and supplemented, individually provided 11 liters of milk in the morning. The total volume was clotted raw immediately after milking, at 32°C, followed traditional Colonial process. One hundred cheeses were produced in five different seasons, each one subjected to seven maturation times, where zero for production day, one, two, three, four, six and eight months of maturation. Samples were weighed in duplicate, measured for water activity (Aw), number of lactic acid bacteria (LAB), lipolytic and proteolytic mesophilic and presence of Listeria spp. Temperature and ambient relative humidity were recorded. All data were subjected to analysis of variance (ANOVA), applied Tukey test, at 5% significance and analyzed by Sisvar statistical program. In the first study, the parameters on the Traditional Colonial Cheese were evaluated from time zero to eighth months of maturation. The lineation was completely randomized, in a double factorial scheme, considering the production period as factor 1 and maturation time as factor 2. The second work evaluated the peeling treatment occurred in the third month, in times four, six and eight months of maturation. The lineation was in randomized blocks in a scheme of split plots, being block: production period; plot: maturation time; and subplot: peeling treatment. In the first work, for weight loss, there was no difference between the cheeses at the end of eight months. The loss was relevant until the second month of maturation, the process initial critical phase. To Aw, cheeses made in May and June remained with the highest concentrations, and the ones produced in November with the lowest values. To BAL, for all assessed cheeses there was significant increase in the first month, but the decrease occurred only for cheeses made in November and May, indicating that the process of acidification and subsequent maturation stabilization may have occurred with more efficiency in these months. As for lipolytic bacteria, cheeses made in May and June had the highest scores, probably due to lower temperatures. For proteolytic bacteria, there was an evident effect of the coldest times on the counts, especially until the second month of maturation. Comparing the peeling treatments, in the second study, there was no effect on weight loss, lipolytic and proteolytic mesophilic bacteria counting. Imbriago cheeses showed contents for Aw and lactic acid bacteria counts significantly higher than Traditional Colonial, starting only in the fourth month of maturation, which may be a consequence of the hydration occurring at the peeling treatment time. It can be concluded that cheeses produced in May can be indicated as the best period for production, both for the colonial as for the Imbriago. Regarding the peeling treatment, the probable rehydration does not represent a health risk / Dois experimentos foram conduzidos com o intuito de identificar a melhor época para produção e tempo de maturação de queijo Colonial Tradicional e Colonial Imbriago no município de Pinhão Paraná. Dez fêmeas bovinas Holandês, mantidas em pastagem e suplementadas, forneceram individualmente 11 litros de leite pela manhã. O volume total foi coagulado cru imediatamente pós ordenha, à 32ºC, seguido processo tradicional do Colonial. Cem queijos foram produzidos em cinco diferentes épocas do ano, cada uma submetida em sete tempos de maturação, onde zero para dia da produção, um, dois, três, quatro, seis e oito meses de maturação. Em duplicata as amostras foram pesadas, mensuradas para atividade da água (Aw), número de bactérias láticas (BAL), mesófilas lipolíticas e proteolíticas e presença de Listeria spp. Foram registradas temperatura e umidade relativa ambiente. Todos os dados foram submetidos à análise de variância (ANOVA), aplicado teste de Tukey, a 5% de significância e analisados pelo programa estatístico Sisvar. No primeiro trabalho foram avaliados os parâmetros sobre o Queijo Colonial Tradicional do tempo zero ao oitavo meses de maturação. O delineamento foi inteiramente casualizado em esquema fatorial duplo, considerando o período de produção como fator 1 e tempo de maturação como fator 2. O segundo trabalho avaliou o tratamento da casca ocorrido no terceiro mês, nos tempos quatro, seis e oito meses de maturação. O delineamento foi em blocos casualizados em esquema de parcelas subdivididas, sendo bloco: período de produção; parcela: tempo de maturação; e sub-parcela: tratamento da casca. No primeiro trabalho, para perda de peso, não houve diferença entre os queijos ao final dos oito meses. A perda foi relevante até o segundo mês de maturação, fase crítica inicial do processo. Para Aw, os queijos produzidos em maio e junho se mantiveram com maiores teores e os produzidos em novembro com os menores valores. Para BAL, para todos os queijos avaliados houve aumento significativo no primeiro mês, mas o decréscimo ocorreu apenas para os queijos produzidos em novembro e maio, indicando que o processo de acidificação e posterior estabilização da maturação pode ter ocorrido com maior eficiência nestes meses. Quanto às bactérias lipolíticas, os queijos produzidos em maio e junho tiveram as maiores contagens, provavelmente em função das menores temperaturas. Para bactérias proteolíticas, houve evidente efeito das épocas mais frias sobre as contagens, principalmente até o segundo mês de maturação. Comparados os tratamentos da casca, no segundo trabalho, não houve efeito sobre perda de peso, contagem de bactérias mesófilas lipolíticas e proteolíticas. Os queijos Imbriago apresentaram teores para Aw e contagem de bactérias ácido láticas significativamente superiores ao Colonial Tradicional, que ocorreu apenas no quarto mês de maturação, que pode ser consequência da hidratação ocorrida no momento do tratamento da casca. Pode-se concluir que os queijos produzidos em maio podem ser representar o melhor período para a produção, tanto para o colonial como para o Imbriago. Quanto ao tratamento da casca, a provável reidratação não representa um risco sanitário