Egg is one of the major food allergens that affects 1.6~3.2% of the infants and young children population. The objective of this study is to reduce the egg white IgE binding ability by lactobacilli or Aspergillus oryzae fermentation. Modifications of egg white proteins during fermentation were analyzed by Ninhydrin method, Ellman method, SDS-PAGE, ELISA, and MALDI-TOF-MS. Tryptone supplementation and acidification are necessary to grow lactobacilli in egg white. Egg whites were fermented by L sanfranciscensis, L. sakei, and L. delbrueckii subsp. delbrueckii individually for 96 h; and Aspergillus oryzae for 120 h. The IgE binding ability of egg white was significantly reduced (~50%) by L. delbrueckii subsp. delbrueckii after 48 h of incubation and almost eliminated by Aspergillus oryzae after 24 h of inoculation. In addition to slight modification of ovomucoid (the dominant egg allergen), no substantial protein degradation was observed during fermentation. / Food Science and Technology

Orientador: Yoon Kil Chang / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-14T14:34:15Z (GMT). No. of bitstreams: 1 Silva_LeomarHackbartda_D.pdf: 2305142 bytes, checksum: 6c8e0a6535a2141daa95d72795481f6b (MD5) Previous issue date: 2009 / Resumo: O consumo de soja e produtos fermentados de soja aumentou consideravelmente nos últimos anos, devido à presença de compostos funcionais como as isoflavonas e os peptídeos, que em determinadas concentrações trazem benefícios à saúde dos consumidores. Este trabalho objetivou avaliar e caracterizar a conversão dos isômeros de isoflavonas glicosiladas à agliconas e alterações das propriedades tecnológicas durante a fermentação da farinha de soja integral autoclavada (FSIA), sendo as condições de fermentação otimizadas através de Metodologia de Superfície de Resposta e posterior aplicação em pão de forma. A FSIA foi fermentada com o fungo Aspergillus oryzae CCT 4359, variando-se a temperatura de 28 (-1,41) a 42ºC (+1,41), e o tempo de incubação de 19 (-1,41) a 53 h (+1,41) de acordo com o planejamento experimental, seca em estufa a vácuo, a 60ºC, até 10% de umidade, obtendo-se a farinha de soja integral autoclavada fermentada (FSIAF), a qual foi avaliada quanto à composição centesimal, índice de absorção de água (IAA), índice de solubilidade do nitrogênio (ISN), índice de atividade ureática (IAU), pH, índice de acidez do extrato etéreo (IAE), cor instrumental, análise quantitativa das isoflavonas (CLAE-DAD) e perfil eletroforético das proteínas (SDS-PAGE). A otimização da formulação de pão de forma com FSIAF-otimizada foi realizada através de um planejamento experimental variando-se a porcentagem de FSIAF e de vital glúten. Os pães foram avaliados quanto ao volume específico, cor do miolo, umidade e firmeza do miolo. O produto otimizado foi caracterizado quanto ao volume específico e cor do miolo no dia da produção, umidade e firmeza do miolo, nos dias 1, 4 e 7 após o processamento, análise sensorial (aparência, cor, aroma, textura e sabor), intenção de compra e quantificação das isoflavonas. Os resultados indicaram que a fermentação aumentou o conteúdo de proteínas, lipídeos, cinzas e fibras, reduzindo o teor de carboidratos e não alterou a umidade. Modificou o IAA e IAE, reduziu o IAU de 0,61 (FSIA) para 0,25 unidades de pH (FSIAF), o que indica a redução de fatores antinutricionais, porém não influenciou na cor, pH e ISN da FSIAF, promoveu a transformação das isoflavonas glicosiladas em agliconas que passaram de 35,13 µg.g-1 na FSIA para a faixa de 245,23 a 535,94 µg.g-1 na FSIAF, nos diferentes ensaios, aumentando em no máximo 14,26 vezes a concentração de agliconas na FSIAF e também promoveu a formação de peptídeos de baixo peso molecular (< 20 kDa). A FSIAF a 35ºC por 36h (ponto central) aumentou as isoflavonas agliconas para 77% do total de isoflavonas, reduziu a atividade ureática para níveis aceitáveis, não alterou os parâmetros de ISN e IAA, mantendo o IAE baixo, quando comparado com a FSIA, obtendo-se uma FSIAF-otimizada com melhores propriedades nutricionais, tecnológicas e funcionais, sendo escolhida para utilização em formulação de pães de forma. A adição de até 25% de FSIAF-otimizada e 6,0% de vital glúten na formulação de pão de forma não interferiu na firmeza e nas características sensoriais do produto, sendo esta porcentagem recomendada para a substituição da farinha de trigo. Uma porção de 50 g de pão de forma com adição de 25% de FSIAF e 6% de vital glúten fornece 10,20 g de proteína total, sendo 3,92 g de proteína de soja, que representam 15,66% da Ingestão Diária Recomendada, com 218,62 µg de isoflavonas, contendo 71% na forma aglicona o que corresponde a 44% da ingestão de isoflavonas considerada para efeito benéfico à saúde (500 µg). Além de fornecer 1,0% de fibras totais, conferindo propriedades funcionais ao pão / Abstract: The consumption of soybean and its fermented products has increased considerably in recent years due to the presence of functional compounds such as isoflavones and peptides, which are of benefit to consumer health if consumed in determined concentrations. The objective of the present study was to evaluate and characterize the conversion of glycosylated isoflavone isomers to aglycones and alterations in the technological properties, during the fermentation of whole autoclaved soybean flour (WASF), the fermentation conditions being optimized using the Response Surface Methodology and subsequent application in pan bread. The WASF was fermented using the fungus Aspergillus oryzae CCT 4359, varying the temperature from 28ºC (-1.41) to 42ºC (+1.41) and the incubation time from 19h (-1.41) to 53h (+1.41) according to the experimental design, and then dried to 10% moisture content in a vacuum oven at 60ºC, thus obtaining whole fermented autoclaved soybean flour (WFASF). The following characteristics were evaluated: proximate composition, water absorption index (WAI), soluble nitrogen index (SNI), ureatic activity index (UAI), pH, ether extract acidity index (EAI), instrumental color, quantitative analysis of the isoflavones (HPLC-PDA) and the electrophoretic profile of the proteins (SDS-PAGE). Optimization of the formulation for the bread loaves containing optimized WFASF was carried out using an experimental design, varying the percentages of WFASF and vital gluten. The specific volume, crumb color, moisture content and firmness of the loaves were evaluated. The optimized product was characterized with respect to specific volume and crumb color on the day of production, the moisture content and firmness 1, 4 and 7 days after processing, also determining the sensory acceptance (appearance, color, aroma, texture and flavor), purchase intention and quantification of the isoflavones. The results indicated that fermentation increased the protein, lipid, ash and fiber contents, reduced the carbohydrate content and did not alter the moisture content. It modified the WAI and EAI, reduced the UAI from 0.61 (WASF) to 0.25 (WFASF) pH units, indicating a reduction in the anti-nutritional factors, but did not influence the color, pH or SNI of the WFASF, promoting transformation of the glycosylated isoflavones into aglycones, which increased from 35.13 µg.g-1 in the WASF to a range between 245.23 to 535.94 µg.g-1 in the WFASF in the different trials, increasing the concentration of aglycones up to 14.26 times in the WFASF as compared to the WASF, and also promoting the formation of low molecular weight peptides (<20 kDa). In the WFASF produced at 35ºC for 36h (central point), the isoflavone aglycones increased to 77% of the total isoflavones, the ureatic activity was reduced to acceptable levels, the parameters SNI and WAI were unaltered, and the EAI remained low, as compared to the WASF, thus obtaining an optimized WFASF with better nutritional, technological and functional properties, allowing for its use in the formulation of bread loaves. The addition of 25% optimized WFASF and 6.0% vital gluten to the bread loaf formulation did not interfere with the firmness or sensory characteristics of the product, this being the proportion recommended for substitution of part of the wheat flour. A 50 g serving of bread with this percentage of addition provided 3.92 g soybean protein, representing 15.66% of the recommended daily ingestion, and 218.62 µg isoflavones with 71% in the aglycone form, corresponding to 44% of the recommended ingestion of isoflavones for a beneficial effect on health (500 µg), as well as providing 1.0% total fiber, thus conferring functional properties to the bread / Doutorado / Doutor em Tecnologia de Alimentos

Xylanase was purified from the crude culture of Aspergillus oryzae DSM1863 by sephadex G200 and DEAE – cellulose ion exchange chromatography. The molecular mass of the purified xylanase determined by SDS–PAGE was 21 kDa with a specific activity of 6768 U/mg towards 1% (w/v) of birch wood xylan. The optimum temperature was observed at 60°C. The enzyme was thermostable in the temperature range of 37-50°C with a high residual activity of 62-74% (650.6- 775.9 U/mg protein). / Enzyme xylanase được tinh sạch từ dịch lên men của chủng Aspergillus oryzae DSM1863 sau khi qua cột sắc ký lọc gel sephadex G200 và sắc ký trao đổi ion DEAE – cellulose. Khối lượng phân tử của enzyme xylanase tinh sạch được xác định bằng điên di đồ SDS- PAGE. Xylanase tinh sạch có kích thước là 21 kDa với hoạt tính đặc hiệu đạt 6768 U/mg sau khi được xác định với nồng độ cơ chất là 1% birch wood xylan. Nhiệt độ tối ưu để enzyme hoạt động mạnh nhất là 60°C. Enzyme xylanase khá bền nhiệt. Hoạt tính của enzyme vẫn còn duy trì 62-74% (hoạt tính đặc hiệu đạt 650.6-775.9 U/mg protein) sau khi 8 giờ ủ ở 37-50°C.

Os micro-organismos que habitam o interior das plantas (endofíticos ou endófitos) tornaram-se foco de interesse por estarem envolvidos na produção de compostos químicos como enzimas, alcalóides, antibióticos, anticancerígenas e diferentes metabólitos. Os ecossistemas de regiões tropicais tem sido alvo de busca de compostos naturais por causa da riqueza de espécies e nichos ecológicos presentes nestas comunidades. O objetivo deste trabalho o isolamento, identificação e a bioprospecção de fungos endofíticos obtidos de Combretum leprosum e a detecção nos extratos de planta e micro-organismos da presença do composto combretastatin (CA4). Folhas, galhos, frutos e raízes de C. leprosum foram coletados de cinco estados dentro da zona de semiárido brasileiro: Bahia, Piauí, Ceará, Paraíba, Rio Grande do Norte. Partes das amostras foram triturados e submetidos à maceração primeiramente em diclorometano, seguidos de tetrahidrofurano e acetona de acordo com Pettit et al.(1987) para possível extração da CA4. Além disso, para avaliação in vitro da atividade citotóxica e antimicrobiana foram realizadas extrações em acetato de etila, clorofórmio e metanol. Foram detectados a possível presença da CA4 em todos os órgãos das plantas extraídos com tetrahidrofurano e as maiores concentrações foram observadas nas folhas. A atividade antitumoral dos extratos vegetais apresentaram as maiores inibições contra carcinoma (ovário IC50 10&micro;g/mL-1, rim IC50 8,7&micro;g/mL-1 e mama IC50 14,1&micro;g/mL-1) e glioma.IC50 13,5&micro;g/mL-1. A outra parte das amostras (folhas, caules e raízes) foram desinfetadas, fragmentadas e colocadas em meios de cultivo (Martin, BDA, Agar água) por 60 dias, 28&deg;C. Foram isolados 405 fungos endofíticos e 159 apresentaram atividade contra fitopatogênicos, 72% para Rhizoctonia solani e 28% para Pythium aphanidermatum. As vinte e três linhagens que apresentaram as melhores atividades antifitopatogênicas foram submetidas a crescimento em Czapec em cultura estacionaria, por 30 dias, a 28&deg;C, os respectivos metabólitos foram obtidos em múltiplo (3.0 e 11.0) e avaliados a atividade antimicrobiana contra bactérias patogênicas e fungos. Quatro linhagens foram selecionadas, identificadas pelo sequenciamento da região 18S, CFE177 como Fusarium oxysporum, CFE03 como Hypocrea koningii, linhagem CFE108 como Aspesgillus oryzae e CFE391 como Fusarium solani e avaliadas in vitro pelos testes biológicos: atividade antitumoral, antioxidante e antimicobactéria. Os compostos produzidos por A. oryzae CFE108 apresentaram potencial para bioprospecção, e de acordo com as atividade citotóxicas as maiores ações foram contra as linhagens linfoma histiocística (J744), mieloma murino (B16F10) e baixa citotóxidade para carcinoma de bexiga (ECV304) e leucemia eritroblástica humana (k562) na concentração de 1mg/mL-1. Foram isolados dois compostos: SS-XL-32-01 identificado como bis-(2-etilhexil) ftalato (DEHP) e SS-XL-20-1 identificado como fenol, 2.2 metilenobis[6-(1,1-dimetiletil)-4- etil], ambos com atividade anticâncer para células HeLa com percentual de ate 98% e 71%, de morte, respectivamente. Alem disso, a modificação através da reação de metilação do composto SS-XL-32-1 resultou na quebra do anel aromático, formação de 4 subprodutos e perda da atividade, sendo um indicativo do sitio ativo da molécula responsável pela atividade observada. Portanto, fungos endofíticos de 18 plantas do semiárido brasileiro podem ser considerados fonte de bioprospecção para novas moléculas bioativas com atividade antitumoral. / The micro-organisms that reside in the aerial tissues and roots of plants (endophytic or endophyte) became the focus of interest for being involved in the chemical production such as enzymes, alkaloids, antibiotics, anticancer and different metabolites. The ecosystems of tropical region have been targeted search of natural compounds because of the richness of species and ecological niches present in these communities. The aim of this work was the isolation, identification and bioprospection for endophytic fungi from Combretum leprosum and detection in extracts of the plant and micro-organisms for the presence of the combretastatin (CA4). Leaves, stems, fruits and roots of C. leprosum were collected from five states within the semi-arid zone of Brazil: Bahia, Piaui, Ceara, Paraiba, Rio Grande do Norte. Part of the samples were crushed and subjected to maceration in dichloromethane, followed by tetrahydrofuran and acetone according to Pettit et al. (1987) for extracting the possible CA4. Moreover, for in vitro evaluation of the cytotoxic and antimicrobial activity extractions were carried out in ethyl acetate, chloroform and methanol. Were detected the possible presence of CA4 all plant organs extracted with tetrahydrofuran and the highest concentrations were observed on the leaves. The antitumor activity of plant extracts showed the highest inhibition against carcinoma (ovary IC50 10&micro;g/mL-1, kidney IC50 8.7 &micro;g/mL-1 and breast IC50 14.1 &micro;g/mL-1) glioma IC50 and 13.5 mg-/mL-1. The other part of the samples (leaves, stems and roots) were disinfected, fragmented and placed in culture media (Martin, PDA, water agar) for 60 days, 28&deg;C. 405 Endophytic fungi were isolated and 159 showed activity against phytopathogenic, 72% for Rhizoctonia solani and 28% for Pythium aphanidermatum. Twenty-three strains that showed good activities antiphytopathogenic, were grow on medium Czapec in static culture, for 30 days at 28&deg;C, the respective metabolites were obtained in multiples pH (3.0 and 11.0) and evaluated the antimicrobial activity against pathogenic bacteria and fungi. Four strains were selected, identified by sequencing the 18S region, CFE177 as Fusarium oxysporum, CFE03 as Hypocrea koningii, strain CFE108 as Aspesgillus oryzae and CFE391 Fusarium solani, and evaluated by in vitro biological tests: antitumor, antioxidant and antimicobactérium activity. The compounds produced by A. oryzae CFE108 had biological potential and in accordance with the cytotoxic activity, showed the highest activities against lymphoma lines (J744), murine myeloma (B16F10) and low cytotoxicity for carcinoma of the bladder (ECV304) and leukemia erythroblastic human (K562) in 1mg/mL-1 concentration. Two compounds were isolated: SS-XL-32- 01 identified as bis-(2-ethylhexyl)phthalate (DEHP), and SS-XL-20-1 as phenol 2.2methylenobis [6-(1,1-dimethylethyl) - 4-ethyl], both with anticancer activity for HeLa cells with a percentage of up to 98% and 71%, of death, respectively. In addition, modified by methylation reaction of the compound SS-XL-32-1 resulted in the breaking of the aromatic ring and result in formation of four product and loss of activity being indicative of the active site of the molecule can be the aromatic ring. Therefore, endophytic fungi in semiarid Brazil plant can be considered a source of bioprospection for new bioactive molecules with anticancer activity.

Os micro-organismos que habitam o interior das plantas (endofíticos ou endófitos) tornaram-se foco de interesse por estarem envolvidos na produção de compostos químicos como enzimas, alcalóides, antibióticos, anticancerígenas e diferentes metabólitos. Os ecossistemas de regiões tropicais tem sido alvo de busca de compostos naturais por causa da riqueza de espécies e nichos ecológicos presentes nestas comunidades. O objetivo deste trabalho o isolamento, identificação e a bioprospecção de fungos endofíticos obtidos de Combretum leprosum e a detecção nos extratos de planta e micro-organismos da presença do composto combretastatin (CA4). Folhas, galhos, frutos e raízes de C. leprosum foram coletados de cinco estados dentro da zona de semiárido brasileiro: Bahia, Piauí, Ceará, Paraíba, Rio Grande do Norte. Partes das amostras foram triturados e submetidos à maceração primeiramente em diclorometano, seguidos de tetrahidrofurano e acetona de acordo com Pettit et al.(1987) para possível extração da CA4. Além disso, para avaliação in vitro da atividade citotóxica e antimicrobiana foram realizadas extrações em acetato de etila, clorofórmio e metanol. Foram detectados a possível presença da CA4 em todos os órgãos das plantas extraídos com tetrahidrofurano e as maiores concentrações foram observadas nas folhas. A atividade antitumoral dos extratos vegetais apresentaram as maiores inibições contra carcinoma (ovário IC50 10&micro;g/mL-1, rim IC50 8,7&micro;g/mL-1 e mama IC50 14,1&micro;g/mL-1) e glioma.IC50 13,5&micro;g/mL-1. A outra parte das amostras (folhas, caules e raízes) foram desinfetadas, fragmentadas e colocadas em meios de cultivo (Martin, BDA, Agar água) por 60 dias, 28&deg;C. Foram isolados 405 fungos endofíticos e 159 apresentaram atividade contra fitopatogênicos, 72% para Rhizoctonia solani e 28% para Pythium aphanidermatum. As vinte e três linhagens que apresentaram as melhores atividades antifitopatogênicas foram submetidas a crescimento em Czapec em cultura estacionaria, por 30 dias, a 28&deg;C, os respectivos metabólitos foram obtidos em múltiplo (3.0 e 11.0) e avaliados a atividade antimicrobiana contra bactérias patogênicas e fungos. Quatro linhagens foram selecionadas, identificadas pelo sequenciamento da região 18S, CFE177 como Fusarium oxysporum, CFE03 como Hypocrea koningii, linhagem CFE108 como Aspesgillus oryzae e CFE391 como Fusarium solani e avaliadas in vitro pelos testes biológicos: atividade antitumoral, antioxidante e antimicobactéria. Os compostos produzidos por A. oryzae CFE108 apresentaram potencial para bioprospecção, e de acordo com as atividade citotóxicas as maiores ações foram contra as linhagens linfoma histiocística (J744), mieloma murino (B16F10) e baixa citotóxidade para carcinoma de bexiga (ECV304) e leucemia eritroblástica humana (k562) na concentração de 1mg/mL-1. Foram isolados dois compostos: SS-XL-32-01 identificado como bis-(2-etilhexil) ftalato (DEHP) e SS-XL-20-1 identificado como fenol, 2.2 metilenobis[6-(1,1-dimetiletil)-4- etil], ambos com atividade anticâncer para células HeLa com percentual de ate 98% e 71%, de morte, respectivamente. Alem disso, a modificação através da reação de metilação do composto SS-XL-32-1 resultou na quebra do anel aromático, formação de 4 subprodutos e perda da atividade, sendo um indicativo do sitio ativo da molécula responsável pela atividade observada. Portanto, fungos endofíticos de 18 plantas do semiárido brasileiro podem ser considerados fonte de bioprospecção para novas moléculas bioativas com atividade antitumoral. / The micro-organisms that reside in the aerial tissues and roots of plants (endophytic or endophyte) became the focus of interest for being involved in the chemical production such as enzymes, alkaloids, antibiotics, anticancer and different metabolites. The ecosystems of tropical region have been targeted search of natural compounds because of the richness of species and ecological niches present in these communities. The aim of this work was the isolation, identification and bioprospection for endophytic fungi from Combretum leprosum and detection in extracts of the plant and micro-organisms for the presence of the combretastatin (CA4). Leaves, stems, fruits and roots of C. leprosum were collected from five states within the semi-arid zone of Brazil: Bahia, Piaui, Ceara, Paraiba, Rio Grande do Norte. Part of the samples were crushed and subjected to maceration in dichloromethane, followed by tetrahydrofuran and acetone according to Pettit et al. (1987) for extracting the possible CA4. Moreover, for in vitro evaluation of the cytotoxic and antimicrobial activity extractions were carried out in ethyl acetate, chloroform and methanol. Were detected the possible presence of CA4 all plant organs extracted with tetrahydrofuran and the highest concentrations were observed on the leaves. The antitumor activity of plant extracts showed the highest inhibition against carcinoma (ovary IC50 10&micro;g/mL-1, kidney IC50 8.7 &micro;g/mL-1 and breast IC50 14.1 &micro;g/mL-1) glioma IC50 and 13.5 mg-/mL-1. The other part of the samples (leaves, stems and roots) were disinfected, fragmented and placed in culture media (Martin, PDA, water agar) for 60 days, 28&deg;C. 405 Endophytic fungi were isolated and 159 showed activity against phytopathogenic, 72% for Rhizoctonia solani and 28% for Pythium aphanidermatum. Twenty-three strains that showed good activities antiphytopathogenic, were grow on medium Czapec in static culture, for 30 days at 28&deg;C, the respective metabolites were obtained in multiples pH (3.0 and 11.0) and evaluated the antimicrobial activity against pathogenic bacteria and fungi. Four strains were selected, identified by sequencing the 18S region, CFE177 as Fusarium oxysporum, CFE03 as Hypocrea koningii, strain CFE108 as Aspesgillus oryzae and CFE391 Fusarium solani, and evaluated by in vitro biological tests: antitumor, antioxidant and antimicobactérium activity. The compounds produced by A. oryzae CFE108 had biological potential and in accordance with the cytotoxic activity, showed the highest activities against lymphoma lines (J744), murine myeloma (B16F10) and low cytotoxicity for carcinoma of the bladder (ECV304) and leukemia erythroblastic human (K562) in 1mg/mL-1 concentration. Two compounds were isolated: SS-XL-32- 01 identified as bis-(2-ethylhexyl)phthalate (DEHP), and SS-XL-20-1 as phenol 2.2methylenobis [6-(1,1-dimethylethyl) - 4-ethyl], both with anticancer activity for HeLa cells with a percentage of up to 98% and 71%, of death, respectively. In addition, modified by methylation reaction of the compound SS-XL-32-1 resulted in the breaking of the aromatic ring and result in formation of four product and loss of activity being indicative of the active site of the molecule can be the aromatic ring. Therefore, endophytic fungi in semiarid Brazil plant can be considered a source of bioprospection for new bioactive molecules with anticancer activity.

Doctor of Philosophy / Department of Grain Science and Industry / Praveen V. Vadlani / Soy meal (SM) is the main protein source for monogastric animals. Anti-nutritional factors in SM limited its usage for young monogastric animals. Aspergillus was investigated to degrade these factors and to enhance its nutritional value via solid state fermentation. Galacto-oligosaccharides were totally degraded from the initial 9.48 mmol/100 g, and trypsin inhibitor decreased from 10.7 TIU/mg to a non-detectable level after 36 hr fermentation. Structural polysaccharides decreased by 59% (w/w) and the degree of hydrolysis of SM protein increased from 0.9% to 7% (w/w) through the 7 d fermentation. Fermentation also modified nutritional factors. Protein content increased from 50.47% (w/w) to 58.93% (w/w) after 36 hr fermentation. Amino acid profile was significantly enhanced. Two - stage temperature-induced fermentation protocol was developed to increase the degradation rate of phytic acid by A. oryzae (ATCC 9362) and by A. ficuum (ATCC 66876). The first stage maximized phytase production with fermentation parameters obtained by central composite design. The second stage achieved maximum enzymatic degradation with parameters obtained by studying the phytase temperature characteristics. While using A. oryzae, 57% of phytic acid in SM was degraded by the two stage protocol compared to 39% degradation from single stage fermentation. For A. ficuum, the two-stage temperature fermentation protocol achieved a 98% degradation level of phytic acid degradation compared with the single stage process. Two-stage temperature-induced co-fermentation of A. oryzae and A. ficuum was investigated to simultaneously degrade phytic acid and soy protein with high efficiency. Co-fermentation of A. oryzae and A. ficuum resulted in higher phytic acid degradation than A. oryzae fermentation and superior protein hydrolysis compared to A. ficuum fermentation. Sterilization distorted the results of fermentation effect on soy allergens and soy protein degradation. Virginiamycin is a kind of bacterisin. It was added to A. oryzae solid state fermentation, to exclude the necessity of SM sterilization. Nonsterile, solid state fermentation using A. oryzae and virginiamycin showed the complete degradation of α and α’ subunits of β-conglycinin and decreased immunoreactivity of soy protein. The modified SM after microbial bioprocessing created an innovative product with enhanced characteristics with potential wider applications for feed industry.

Computational tools play an important role in the description of biological systems. Scientists describe and study structure, conformational changes and interactions between molecules in silico, often as a cheaper and faster alternative for biosynthesis. The simulated dynamic behavior in time of a molecular system is a straight forward source of information about substrate-enzyme interactions at the atomic level, and a powerful tool for the identification of molecular properties important in enzymatic reactions. Our study is focused on the computational investigation of structure and substrate specificity of hydrolases important in biotransformation. The computational work was performed in close collaboration with biochemists-experimentalists from Charles University and the Microbiological Institute of the Academy of Sciences of the Czech Republic. Hydrolases have great a potential in the chemoenzymatic synthesis of modified carbohydrates with regulated properties. Carbohydrates, as substrates of hydrolases, are important in normal functionality of many organisms. They have a dual role in immune response regulation: some carbohydrates (like GlcNAc and ManNAc) participate in activation and some (like GalNAc) in suppressing immunity; glycosidase deficiency is associated with a number of lysosomal disorders. We used homology modeling, computational docking and molecular dynamics simulation (MD) methods for the complex study of fungal hydrolases: alpha-galactosidase/alpha-N-acetylgalactosaminidase from Aspergillus niger; beta-N-acetylhexosaminidases (HEX) (from Aspergillus oryzae and Penicillium oxalicum); nitrilase from Aspergillus niger. Our structural study unambigously demonstrates that the enzyme encoded by genes variant A (aglA) from A. niger is able to accept alpha-N-acetylgalactosamine as its substrate and explains structural features responsible for its specificity. Homology models of HEXs from P. oxalicum and A. oryzae were built and compared. Homology models were used to study the role of protein glycosylation, disulfide bonds, dimer formation and interaction with natural and modified substrates. Model of nitrilase from Aspergillus niger helped to analyze multimer formation.

京都大学 / 0048 / 新制・論文博士 / 博士(農学) / 乙第12763号 / 論農博第2786号 / 新制||農||1016(附属図書館) / 学位論文||H25||N4786(農学部図書室) / 30615 / (主査)教授 平竹 潤, 教授 植田 充美, 教授 小川 順 / 学位規則第4条第2項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

Submitted by isabela.moljf@hotmail.com (isabela.moljf@hotmail.com) on 2017-08-22T16:13:10Z No. of bitstreams: 1 miguelgontijosiqueirapalmieri.pdf: 16600771 bytes, checksum: 1474cea0351558e2cfb2fcb3834f5ff7 (MD5) / Rejected by Adriana Oliveira (adriana.oliveira@ufjf.edu.br), reason: on 2017-08-24T11:35:30Z (GMT) / Submitted by isabela.moljf@hotmail.com (isabela.moljf@hotmail.com) on 2017-08-24T13:34:54Z No. of bitstreams: 0 / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2017-08-30T12:31:36Z (GMT) No. of bitstreams: 0 / Made available in DSpace on 2017-08-30T12:31:36Z (GMT). No. of bitstreams: 0 Previous issue date: 2017-02-03 / O café verde é conhecido principalmente por suas propriedades antioxidantes e antibacterianas. Compostos fenólicos como os ácidos clorogênicos, que são formados através de ácidos hidroxicinâmicos (ácidos cafeico, ferúlico e outros) ligados ao ácido quínico, estão presentes em grandes quantidades e têm sido reconhecidos como antioxidantes naturais. Entretanto, os ácidos hidroxicinâmicos são encontrados principalmente na forma esterificada com ácidos orgânicos, açúcares, lipídeos e covalentemente ligados a parede celular, o que reduz sua biodisponibilidade. Diante disso, o objetivo deste estudo foi realizar a biotransformação da farinha de café verde utilizando o fungo Aspergillus oryzae (CCDCA102604) visando o aumento da atividade antioxidante. A farinha de café verde foi fermentada em estado sólido usando o fungo Aspergillus oryzae a 25 °C por 24, 48 e 72 horas. Após a fermentação os compostos fenólicos foram extraídos utilizando uma solução hidroetanólica. A atividade antioxidante dos extratos foi avaliada pelos métodos DPIDI-1• (2,2-difenil-1-picril-hidrazila) e poder redutor e a determinação dos teores de fenólicos totais foi realizada utilizando o método de Folin-ciocateu. Foi realizada também a quantificação de ácido clorogênico (5-ACQ), ácido cafeico e cafeína nos extratos utilizando a cromatografia líquida de alta eficiência. De acordo com os resultados ocorreu um aumento significativo de 115,7% e 66,4% da atividade antioxidante dos extratos de farinha de café verde fermentados por 24 horas em relação ao extrato de café não fermentado, determinada pelos métodos DPPI-1• e poder redutor, respectivamente. Além disso, o processo de fermentação pelo fungo A. oryzae durante 24 horas também promoveu um aumento de 68,6% na concentração de compostos fenólicos em relação aos extratos não fermentados. Os extratos fermentados por 24 horas apresentaram um aumento significativo nos teores de ácido clorogênico e ácido cafeico quando comparados aos extratos não biotransformados. O aumento da atividade antioxidante não foi observado nos extratos fermentados por 48 e 72 horas. Os resultados deste estudo demostraram que o processo de biotransformação é uma estratégia para a obtenção de um extrato enriquecido de compostos antioxidantes em suas formas livres com potencial aplicação nas indústrias alimentícias, de suplementos alimentares e cosmética. / Green coffee is known mainly for its antioxidant and antibacterial properties. Phenolic compounds such as chlorogenic acids, which are formed through hydroxycinnamic acids (caffeic, ferulic and other acids) linked to quinic acid, are present in large quantities in green coffee and have been recognized as natural antioxidants. However, hydroxycinnamic acids are found mainly in the esterified form with organic acids, sugars, lipids and are covalently bound to the cell wall, which reduces their bioavailability. Therefore, the objective of this study was to perform the biotransformation of the green coffee flour using the Aspergillus oryzae fungus aimed at increasing the antioxidant activity. The green coffee flour was fermented in solid state using the fungus Aspergillus oryzae (CCDCA102604) at 25 °C for 24, 48 and 72 hours and after fermentation the phenolic compounds were extracted using a hydroethanolic solution. The antioxidant activity of the extracts was evaluated by the DPPH• and reducing power methods, and the determination of total phenolic contents was performed using Folin-ciocateu method. Quantification of chlorogenic acid (5-ACQ), caffeic acid and caffeine in the extracts was also performed using high performance liquid chromatography. The results showed a significant increase of 115.7% and 66.4% of the antioxidant activity of the green coffee flour extracts fermented for 24 hours in relation to the unfermented coffee extract, measured by the DPPI-1• and reducing power methods, respectively. In addition, the fermentation process by the A. oryzae fungus for 24 hours also promoted a 68.6% increase in the content of phenolic compounds in relation to the unfermented extracts. The extracts fermented for 24 hours showed an increase in the content of chlorogenic acid and caffeic acid when compared to the non-biotransformed extracts. The increase in antioxidant activity was not observed in the extracts fermented for 48 and 72 hours. The results of this study showed the biotransformation process as a strategy to obtain an enriched extract of antioxidant compounds in their free forms with potential application in the food, supplementation and cosmetic industries.

Doctor of Philosophy / Department of Grain Science and Industry / Praveen V. Vadlani / The purpose of this study was to investigate micro- and macro-scale aspects of solid state fermentation (SSF) for production of cellulolytic enzymes using fungal cultures. Included in the objectives were investigation of effect of physicochemical characteristics of substrate on enzymes production at micro-scale, and design, fabrication and analysis of solid-state bioreactor at macro-scale. In the initial studies response surface optimization of SSF of soybeans hulls using mixed culture of Trichoderma reesei and Aspergillus oryzae was carried out to standardize the process. Optimum temperature, moisture and pH of 30ºC, 70% and 5 were determined following optimization. Using optimized parameters laboratory scale-up in static tray fermenter was performed that resulted in production of complete and balanced cellulolytic enzyme system. The balanced enzyme system had required 1:1 ratio of filter paper and beta-glucosidase units. This complete and balanced enzyme system was shown to be effective in the hydrolysis of wheat straw to sugars. Mild pretreatments– steam, acid and alkali were performed to vary physicochemical characteristics of soybean hulls – bed porosity, crystallinity and volumetric specific surface. Mild nature of pretreatments minimized the compositional changes of substrate. It was explicitly shown that more porous and crystalline steam pretreated soybean hulls significantly improved cellulolytic enzymes production in T. reesei culture, with no effect on xylanase. In A. oryzae and mixed culture this improvement, though, was not seen. Further studies using standard crystalline substrates and substrates with varying bed porosity confirmed that effect of physicochemical characteristics was selective with respect to fungal species and cellulolytic activity. A novel deep bed bioreactor was designed and fabricated to address scale-up issues. Bioreactor’s unique design of outer wire mesh frame with internal air distribution and a near saturation environment within cabinet resulted in enhanced heat transfer with minimum moisture loss. Enzyme production was faster and leveled within 48 h of operation compared to 96 h required in static tray. A two phase heat and mass transfer model was written that accurately predicted the experimental temperature profile. Simulations also showed that bioreactor operation was more sensitive to changes in cabinet temperature and mass flow rate of distributor air than air temperature.