Stress constitutes a more and more common cause for many health disorders inmodern society. Salivary -amylase (AA), the most abundant enzyme in humanwhole saliva, has in recent years been found to be a good surrogate biomarker formonitoring stress levels in individuals. This work aims to form the foundation ofa novel approach for measuring the activity of the enzyme in saliva samples bymeans of electrochemistry. The idea is to implement several enzymes along witha starch substrate and an electron mediator in a single system. This system isthen to be coated onto a screen-printed electrode (SPE), which is used along withan electrical component, designed to give rise to a quantifiable, electrical signalwhen the starch is broken down by the AA contained in an added saliva sample.Several such enzyme systems are here qualitatively evaluated. As electron mediator,ferro-/ferricyanide is used. Two different enzymes, glucose oxidase (GOx) andpyrroloquinoline quinone dependent glucose dehydrogenase (PQQ-GDH), are testedfor making up the saccharide oxidising part of the system. Both prove themselvescapable in terms of qualitatively giving rise to an electrical signal. But, in terms ofinternal quantitative comparisons between the two, no practical experiments areperformed in this work. In some runs, the enzyme -glucosidase (AG) is used asan intermediate for breaking down the AA/starch oligosaccharide products intomonosaccharides. This to increase the system’s electrical signal output when usingGOx as oxidising agent. Regrettably, due to lack of AG enzyme, these runs do notprovide any conclusive data, and so further investigations of systems including thisenzyme are needed. Otherwise, all systems tested seem to work, and neither ofthem appear better than the others. Therefore, all of them will require furtherquantitative testing to determine which one is best to implement in the final designof the enzyme system to be applied onto the SPE.

Biological fluids, like saliva, are commonly encountered in forensic casework. The ability to locate and identify the type of biological fluid on a piece of evidence can lead to further testing including DNA extraction and analysis. Saliva stains are often found on a variety of surfaces in the presence of additional bodily fluids. Many of these stains cannot be readily seen, which makes detection difficult. A study utilizing mapping with Seratec® Amylase Paper and the use of an alternative light source (ALS) for better visibility and detection was conducted to test the effectiveness of this medium. Five different types of stains were prepared, including saliva, saliva and blood, saliva and semen, saliva and urine, and saliva, blood, semen, and urine. The stains were deposited onto four different textile types, including cotton, denim, fleece, and spandex. The results indicated that the presence of other body fluids may adversely affect the detection of saliva using Seratec® Amylase Paper. In order to effectively test whether the Amylase Paper itself inhibited DNA extraction and quantification, only half of each saliva stain was mapped with the paper. This left half of the stain untouched, and available for a comparative DNA study. The same saliva donor, donor C, was used for the entirety of the DNA study, and stains were extracted from the cotton and fleece textiles. A Harris micro-punch was used to collect identical 3mm samples from the portion of the stain in contact with the Amylase Paper and the portion of the same stain not in contact with the Amylase Paper. In addition, samples of the Amylase Paper that had not been used for previous testing were tested to see if the internal positive control (IPC) was affected by the paper itself. The results of the DNA extraction and quantification showed no inhibition in the samples in contact with the Amylase Paper, the samples not in contact with the Amylase Paper, nor from the Amylase Paper itself. These results show that Seratec® Amylase Paper can identify saliva in most mixed samples including blood, semen, and urine. In addition, the application of the Amylase Paper does not inhibit or prevent the subsequent extraction or quantification of DNA, and allows for samples in contact with Amylase Paper to be used for DNA testing downstream. Seratec® Amylase Paper is an effective screening method in forensic casework when the presence of saliva is suspected and can be used even when DNA testing is anticipated in the future.

The texture of tortillas was improved by the addition of maltogenic amylase and carboxymethylcellulose (CMC) and guar gum to fresh masa from ground nixtamal (FNM) and nixtamalized corn flour (NCF) masa. Differences in the performance of additives in tortillas held under refrigeration or ambient storage were documented. For NCF tortillas, significant improvements were obtained in objective and subjective texture measurements by two treatments. Tortilla texture was improved by a treatment with a high enzyme level (170 mg/kg of maltogenic α-amylase, 0.14% CMC, 0.85% guar) as measured by objective tests and by a treatment with low enzyme level (60 mg/kg of maltogenic α-amylase, 0.43% CMC, 0.57% guar) as measured by subjective tests. The addition of maltogenic α-amylase (70 mg/kg) and CMC (0.35%) to FNM tortillas at levels similar to the low enzyme NCF treatment but with lower guar level (0.12%) improved tortilla texture. The maltogenic α-amylase softened tortillas by trimming the starch structure. This allowed the guar to interfere with amylopectin re-crystallization inside gelatinized starch granules. The CMC created a more flexible intergranular matrix that helped maintain the disrupted tortilla structure. Guar was ineffective in refrigerated tortillas, whereas, maltodextrins effectively improved refrigerated tortillas. The sequence of partial starch hydrolysis, warm holding condition, and time for guar to associate with starch and CMC was necessary to improve tortilla texture. Thus, different additives may be required for cold versus room temperature storage. Sugars increased in enzyme-treated tortillas during storage. This suggests that maltogenic α-amylase was only partially inactivated during baking of corn tortillas. Tortillas with more enzyme had lower and later pasting viscosity as measured by a Rapid Viscoanalyzer. Tortillas prepared from FNM also had lower and later pasting viscosity compared to NCF tortillas. Pasting viscosity of tortillas revealed intrinsic starch polymer characteristics and interactions. Results of this study provide commercially applicable information about desired levels for the extent of starch hydrolysis, the type and amount of gums and starches, and product microstructure to delay staling of corn tortillas.

The texture of tortillas was improved by the addition of maltogenic amylase and carboxymethylcellulose (CMC) and guar gum to fresh masa from ground nixtamal (FNM) and nixtamalized corn flour (NCF) masa. Differences in the performance of additives in tortillas held under refrigeration or ambient storage were documented. For NCF tortillas, significant improvements were obtained in objective and subjective texture measurements by two treatments. Tortilla texture was improved by a treatment with a high enzyme level (170 mg/kg of maltogenic α-amylase, 0.14% CMC, 0.85% guar) as measured by objective tests and by a treatment with low enzyme level (60 mg/kg of maltogenic α-amylase, 0.43% CMC, 0.57% guar) as measured by subjective tests. The addition of maltogenic α-amylase (70 mg/kg) and CMC (0.35%) to FNM tortillas at levels similar to the low enzyme NCF treatment but with lower guar level (0.12%) improved tortilla texture. The maltogenic α-amylase softened tortillas by trimming the starch structure. This allowed the guar to interfere with amylopectin re-crystallization inside gelatinized starch granules. The CMC created a more flexible intergranular matrix that helped maintain the disrupted tortilla structure. Guar was ineffective in refrigerated tortillas, whereas, maltodextrins effectively improved refrigerated tortillas. The sequence of partial starch hydrolysis, warm holding condition, and time for guar to associate with starch and CMC was necessary to improve tortilla texture. Thus, different additives may be required for cold versus room temperature storage. Sugars increased in enzyme-treated tortillas during storage. This suggests that maltogenic α-amylase was only partially inactivated during baking of corn tortillas. Tortillas with more enzyme had lower and later pasting viscosity as measured by a Rapid Viscoanalyzer. Tortillas prepared from FNM also had lower and later pasting viscosity compared to NCF tortillas. Pasting viscosity of tortillas revealed intrinsic starch polymer characteristics and interactions. Results of this study provide commercially applicable information about desired levels for the extent of starch hydrolysis, the type and amount of gums and starches, and product microstructure to delay staling of corn tortillas.

Antistaling properties of enzymes (xylanase, bacterial maltogenic and conventional a-amylases), CMC and vital wheat gluten on corn tortillas were evaluated during storage for up to 21 days. Effect of storage time (0-21 days) and temperature (-40, -20, 3, 10 and 21 oC) on tortilla staling was evaluated with or without additives. Addition of 275-1650 AU of ICS maltogenic amylase effectively reduced amylopectin retrogradation without reducing tortilla yields, but did not improve tortilla flexibility. The combination of 825 AU of ICS amylase (to interfere with intra-granular amylopectin re-crystallization) and 0.25% CMC (to create a more flexible inter-granular matrix than retrograded amylose) produced less stiff, equally flexible and less chewy tortillas than 0.5% CMC. Corn tortilla staling followed the basic laws that control aging in starch-based semi-crystalline systems such as starch gels, bread and other baked products. Amylopectin re-crystallization was the driving force behind the staling of corn tortillas. Increasing levels of re-crystallized amylopectin measured by DSC correlated significantly with increased tortilla stiffness and reduction in tortilla rollability, pliability and rupture distance during storage. Re-crystallization of amylopectin in fresh tortillas was not detected. It increased rapidly during the first 24 hr reaching a plateau after 7 days storage. The level of amylopectin re-crystallization on tortillas showed a bell-shaped trend along the evaluated storage temperature range with a maximum around 7 oC. However, a negative linear relationship of peak pasting viscosity with storage temperature of tortilla extracts without additives after 21 days suggests other compounds besides amylopectin affect tortilla staling. Thus, interfering with amylopectin re-crystallization is not the only way to retard staling. Further research is required to optimize the addition of maltogenic amylases in continuous processing lines that use fresh masa instead of nixtamalized corn flour, to determine how these amylases interfere with amylopectin re-crystallization and to elucidate if amylose retrogradation continues during storage and plays a role in tortilla staling.

Typescript. / Thesis (Ph. D.)--University of Hawaii, 1968. / Bibliography: leaves 144-149. / xiv, 149 l illus., tables

Universiẗat, Diss., 2002--Göttingen.

Orientador: Rubens Monti / Banca: Edwil Aparecida de Lucca Gattás / Banca: Adriane Maria Ferreira Milagres / Banca: Raquel de Lima Camargo Giordano / Banca: Benevides Costa Chitunda Pessela / Resumo: Neste trabalho estudou-se a possibilidade de imobilização de uma amilase produzida por cepa de Neurospora crassa (mutante exo-1), e produção de derivados ativos e estabilizados. Foram testados diferentes suportes sólidos, incluindo-se diferentes suportes de agarose e suportes epóxidos preparados com Eupergit e Sepabeads. Além da agarose 10BCL foi utilizada a agarose 4BCL para que se verificasse possíveis dificuldades difusionais do substrato desta enzima, o amido. O acompanhamento das cinéticas de imobilização com a maltose como alternativa ao amido, também colaborou em evidenciar a dificuldade de difusão do amido através de ambos os suportes glioxil-agarose. O derivado obtido com agarose 10BCL, assim como aquele produzido com uso de suporte Eupergit foram os derivados mais estáveis, capazes de manter 100% de suas atividades após 12 horas de incubação à temperatura de 60°C, quando na forma solúvel a enzima conservou apenas 12% de sua atividade inicial. Quando incubados a 70°C destacou-se o derivado de glioxil-agarose (10BCL) como mais estável, mantendo cerca de 30% de sua atividade inicial após 4 horas de incubação. Quando testada a utilização de uma agarose comercial alternativa, sem percentual de crosslink conhecido, de menor custo, sua aplicação mostrou-se promissora e os derivados produzidos além de ativos se apresentam estáveis frente à temperatura. Em conjunto, as informações contidas no presente estudo indicam que a amilase de Neurospora crassa apresenta-se promissora em comparação às amilases de mercado aqui estudadas, tanto em sua utilização na forma solúvel quanto no que se relaciona a produção de derivados estáveis. / Abstract: In this work were studied the immobilization of amylase from Neurospora crassa (Mutant Exo-1). This amylase showed easily production, purification and high capacity of immobilization on agarose and epoxy supports. It was used crosslinked agarose with two polymer concentration: 4 and 10%. The 4BCL agarose presents bigger porous diameter than 10 BCL agarose, so, in this case, possible diffusion problems of the starch across the supports could be reduced. Also, in this study, we have tested two epoxy supports for this amylase immobilization, using Eupergit and Sephabeads supports. The activies of the obtained derivatives were measured using two substrates - maltose and starch. Both glyoxyl agarose support prepared with 4BCl and 10BCL agarose present diffusion problems when the starch was used as substrate to measure the immobilization course. The 10BCL glyoxyl derivative presented the highest thermal stability when comparing the others derivatives. Among the epoxy derivatives the Eupergit one were better than the derivatives obtained using Sepabeads as support. In a confrontation between the two best derivatives, that is, the glyoxyl 10 BCL and Eupergit derivatives, both of them were stable at 60º incubation, maintaining 100% of activities for 12 hours, while the soluble amylase preserved about 12% of initial activity. These two amylase derivatives only showed differences at 70ºC incubation, when the glyoxyl 10BCL amylase derivative was more thermally stable, preserving about 30% of the initial activity after 4 hours. / Doutor

Orientador: Gisela Ferreira / Banca: Carmen Silvia Fernandes Boaro / Banca: Ana Catarina Cataneo / Banca: Sônia Cristina Juliano Gualiere de Andrade Perez / Banca: José Antonio Proença Vieira de Moraes / Resumo: O interesse pelo maracujazeiro-doce (Passiflora alata Curtis) tem crescido devido à qualidade dos frutos para consumo in natura e aos preços alcançados no mercado de frutas frescas. Iniciativas de expansão do cultivo de maracujazeiro-doce são de grande importância tanto para o mercado interno quanto para o externo. No entanto, muitos são os relatos de baixo percentual de germinação de sementes dessa espécie, o que dificulta a formação de mudas e de porta-enxertos vigorosos e, consequentemente, a formação de pomar uniforme e com boa produção. Isso sugere que trabalhos que estudem o metabolismo germinativo de sementes de maracujazeiro-doce sejam de grande importância. Sendo assim, foram instalados dois experimentos no Departamento de Botânica e um experimento no Departamento de Química e Bioquímica do Instituto de Biociências, da Unesp, Botucatu, SP com a finalidade de atingir os objetivos propostos. O primeiro experimento constou em determinar as fases da germinação das sementes de P. alata submetidas a concentrações de GA3 e GA4+7 + N-(fenilmetil)-aminopurina, enquanto que o segundo experimento estudou os efeitos desses reguladores vegetais na germinação do maracujá-doce. O delineamento experimental dos dois experimentos foi inteiramente casualizado, com 11 tratamentos e cinco repetições de 25 sementes por parcela, seguindo esquema fatorial 2x5 (reguladores x concentrações) e uma testemunha (água destilada) em comum. As concentrações utilizadas foram 100, 200, 300, 400 e 500 mg L-1 tanto para GA3 quanto para GA4+7 + N-(fenilmetil)- aminopurina. As sementes foram acondicionadas em 'gerbox' preto, contendo 10 mL das soluções com os reguladores vegetais e levadas à câmara de germinação, com temperatura alternada entre 20ºC por 8 h e 30ºC por 16 h. Para o experimento um foram avaliados grau de umidade das sementes, germinabilidade, velocidade e ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Interest in sweet passion fruit (Passiflora alata Curtis) has grown due to the quality of fruits for consumption in natura and prices achieved in the fresh fruit. Initiatives to expand the cultivation of sweet passion fruit are of great importance both for the domestic market and for the external. However there are many data of low percentage of seeds germination of this species. At this way, there is difficulty in the seedlings formation and vigorous rootstock and, consequently, in uniform orchard formation and with good production. This suggests that works to study the metabolism of sweet passion fruit seed germination have great importance. So, it had been installed two experiments at Department of Botany and one experiment at Department of Chemistry and Biochemistry, Bioscience Institute, in São Paulo State University, Botucatu, SP, with the aim to reach the considered purpose data. The first one had consisted of the determination of seeds germination phases subjected to concentrations of GA3 and GA4+7 + N-(phenylmethyl)-1H-purine-6-amine. The second experiment studied the effects of plant growth regulators on sweet passion-fruit germination. The experimental design had been totally randomized, with 11 treatments and five replications of 25 seeds in each plot, following factorial design 2 x 5 (regulators x concentrations), and a common control (distilled water). The seeds had been submitted to the following treatments: 100, 200, 300, 400 and 500 mg L-1, for GA3 and also for GA4+7 + N- (phenylmethyl)-1H-purine-6-amine. The seeds had been sown in black containers, containing 10 mL of solutions with plant growth regulators, and taken to the germination chamber, with temperature alternated between 20ºC for 8 hours and 30º for 16 hours. The seeds moisture degree (%), the percentage of germination, average time of germination, average speed of germination and total percentage of ... (Complete abstract click electronic access below) / Doutor

Orientador: Márcia Rossini Justino Mutton / Banca: Eduardo da Silva Martins / Banca: João Martins Pizauro Júnior / Resumo: A utilização de fermentação em estado sólido a partir de subprodutos agroindustriais possibilita a obtenção de várias biomoléculas de interesse industrial. As amilases são enzimas com grande aplicação nas indústrias têxtil, farmacêuticas, alimentícias e sucroalcooleira, representando aproximadamente 25% do mercado mundial. Neste trabalho foram isoladas três linhagens fúngicas (Rhizopus oryzae, Malbranchea pulchella e Chrysosporium zonatum), que apresentaram potencial amilolítico, quando cultivadas em três resíduos agroindustriais (quirera de arroz e de milho e farelo de trigo). Para a cepa que apresentou maior atividade enzimática, avaliou-se melhor substrato, tempo de cultivo, teores de umidade, fontes suplementares de nitrogênio, pH e temperatura de incubação, objetivando otimizar as condições de cultivo. Observou-se que a maior atividade enzimática foi obtida com a cepa de R. oryzae em 24 horas de fermentação, em meio de cultura contendo farelo de trigo como substrato, em temperatura de 35°C, utilizando solução de sais composta de NH4NO3 a 0,1% e MgSO4.7H2O a 0,1% como fonte de nitrogênio. Alterações de pH entre 4,0 e 6,0 não afetaram significativamente a síntese da enzima. A condições ótimas de atuação foram temperatura de 75°C e pH de 4,5. As enzima manteve-se estável a 75°C na ausência de substrato por 25 minutos / Abstract: The use of fermentation in a solid state from agroindustrial byproducts permits the obtaining of several biomolecules of interest the industry, like the enzymes. The amylases are enzymes with great application on the textile, pharmaceutical and food industries, representing around 25% of the worldwide market. In this work three ungal strains (Rhizopus oryzae, Malbranchea pulchella and Chrysosporium zonatum) were isolated. They presented amylolytic potential when cultivated in three agroindustrial by products (brewers rice, corn grits and wheat bran). To the strain that presented the greatest enzyme activity, it was evaluated the best substrate, cultivation time, moisture content, additional sources of nitrogen, pH and incubation temperature, aiming the optimization of cultivation conditions. I was observed that the greatest enzyme's activity was obtained with 24 hours of fermentation, in R. orizae in a culture medium that contained wheat bran as the substrate, at 35°C, using salt solution made with NH4NO3 0.1%, MgSO4.7h2O 0.1% and (NH4)2SO4 0.1% as nitrogen source. pH alterations between 4.0 and 6.0 didn't change significantly the enzyme's activity. The optimum conditions of performance of the enzyme were temperature of 75°C and pH 4.5. The enzyme kept stable in the lack of substrate for 25 minutes in 75°C / Mestre