by Sham Lok To. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 143-171). / Abstracts in English and Chinese. / Abstract --- p.i / 摘要 --- p.iii / Acknowledgement --- p.iv / Abbreviations --- p.v / Table of contents --- p.vi / List of figures --- p.ix / List of tables --- p.xi / Chapter Chapter One --- Literature Review --- p.1 / Chapter 1.1 --- Introduction --- p.1 / Chapter 1.1.1 --- About L. edodes --- p.3 / Chapter 1.1.2 --- Nutritional and medicinal values of L. edodes --- p.4 / Chapter 1.1.3 --- Life cycle of L. edodes --- p.6 / Chapter 1.1.4 --- Environmental factors affecting fruiting body formation in L. edodes --- p.6 / Chapter 1.2 --- Molecular mechanisms of fruiting body development in L. edodes --- p.8 / Chapter 1.2.1 --- Expression profiling and identification of differentially expressed genes during fruiting --- p.8 / Chapter 1.2.2 --- Changing in membrane structure --- p.11 / Chapter 1.2.3 --- The signal transduction cascade --- p.12 / Chapter 1.3 --- Transformation in L. edodes and in other fungi --- p.14 / Chapter 1.3.1 --- Transformation of L. edodes --- p.14 / Chapter 1.3.2 --- Transformation in other fungi --- p.17 / Chapter 1.4 --- Bioinformatics tools for comparative promoter analysis --- p.22 / Chapter 1.5 --- Objectives and significance --- p.26 / Chapter Chapter Two --- Promoter analysis of differentially expressed genes (DEGs) in the fruiting body development in L. edodes --- p.27 / Chapter 2.1 --- Introduction --- p.27 / Chapter 2.2 --- Materials and methods --- p.29 / Chapter 2.2.1 --- Strains and cultivation conditions --- p.29 / Chapter 2.2.2 --- Genome walking of the 5' flanking region of the DEGs --- p.29 / Chapter 2.2.3 --- Annealing Control Primed (ACP) PCR --- p.31 / Chapter 2.2.4 --- Construction of genomic DNA library --- p.36 / Chapter 2.2.5 --- Nested PCR to amplify the target sequences --- p.37 / Chapter 2.2.6 --- Cloning and sequencing of the 5' flanking region --- p.38 / Chapter 2.2.7 --- Determination of transcription start site by the Neural Network algorithm --- p.39 / Chapter 2.2.8 --- Identification of putative transcription factor binding sites --- p.40 / Chapter 2.3 --- Results --- p.41 / Chapter 2.3.1 --- Construction of adaptor linked template for genome walking --- p.41 / Chapter 2.3.2 --- Sequence analysis and quality control --- p.41 / Chapter 2.3.3 --- Comparison of various methods in genome walking --- p.42 / Chapter 2.3.4 --- Promoter analysis --- p.42 / Chapter 2.4 --- Discussion --- p.58 / Chapter Chapter Three --- In-silico analysis of transcription factor binding sites and identification transcription factors expressed in L. edodes --- p.64 / Chapter 3.1 --- Introduction --- p.64 / Chapter 3.2 --- Material and methods --- p.67 / Chapter 3.2.1 --- Sequence manipulation and extraction of homologous ESTs from C. cinereus --- p.67 / Chapter 3.2.2 --- Extraction of 5' flanking region of the corresponding ESTs and promoter prediction --- p.67 / Chapter 3.2.3 --- Positional cloning of mating type factor A --- p.68 / Chapter 3.3 --- Results --- p.70 / Chapter 3.3.1 --- Sequence extraction and manipulation --- p.70 / Chapter 3.3.2 --- In-silico analysis of transcription factor binding sites in C. cinereus . --- p.70 / Chapter 3.3.3 --- Comparison of putative TFBS between L. edodes and C. cinereus --- p.71 / Chapter 3.3.4 --- Identification of transcription factors in L. edodes by positional cloning --- p.71 / Chapter 3.4 --- Discussion --- p.85 / Chapter Chapter Four --- Identification，expression profiling and promoter analysis of hydrophobin genes --- p.91 / Chapter 4.1 --- Introduction --- p.91 / Chapter 4.2 --- Material and methods --- p.92 / Chapter 4.2.1 --- Clustering and grouping of the hydrophobin ESTs --- p.92 / Chapter 4.2.2 --- Identification of the consensus sequences of the hydrophobin groups --- p.93 / Chapter 4.2.3 --- RNA Sources and Preparation --- p.93 / Chapter 4.2.4 --- Expression profiling of hydrophobin genes by RT-PCR --- p.95 / Chapter 4.2.5 --- Promoter cloning and analysis of hydrophobin genes --- p.95 / Chapter 4.3 --- Results --- p.97 / Chapter 4.3.1 --- Isolation and characterization of four newly found hydrophobin genes --- p.97 / Chapter 4.3.2 --- Expression levels of hydrophobins --- p.100 / Chapter 4.3.3 --- Promoter sequencing of the hydrophobins --- p.103 / Chapter 4.4 --- Discussion --- p.103 / Chapter Chapter Five --- Transformation of L. edodes --- p.110 / Chapter 5.1 --- Introduction --- p.110 / Chapter 5.2 --- Materials and methods --- p.112 / Chapter 5.2.1 --- Vectors and primers design --- p.112 / Chapter 5.2.2 --- Maxi-preparation of plasmids --- p.112 / Chapter 5.2.3 --- Cultural condition and optimization of protoplasts release --- p.114 / Chapter 5.2.4 --- PEG mediated transformation --- p.115 / Chapter 5.2.5 --- Electroporation mediated transformation --- p.116 / Chapter 5.2.6 --- PCR screening of regenerated transformant --- p.116 / Chapter 5.2.7 --- Particle bombardment --- p.117 / Chapter 5.3 --- Results --- p.121 / Chapter 5.4 --- Discussion --- p.128 / Chapter Chapter Six --- General discussions --- p.132 / References --- p.143

Bian Xue Lin. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (p. 189-208). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web.

Development in shiitake mushroom, Lentinula edodes, is a unique process and studies of the molecular basis of this process may lead to improvement in mushroom cultivation. Previous studies have identified a number of signal transduction genes related to mushroom development, but those genes have not been well characterized. The present work characterized a developmentally regulated MAP kinase, Le.MAPK, Histidine kinase Le.nik1 and their interacting partners in the signal transduction pathways. / Histidine kinase Le.nik1 is the first Histidine kinase gene found in basidiomyces by differential display by RAP-PCR and it has a high sequence homology with the Histidine kinase from C. albicans and B. cinerea, which may be related to development and stress responses. A 7.8kb genomic DNA sequence and the full-length ORF of 6.29kb cDNA sequence of the two-component histidine kinase Le.nik1 has been determined. Northern blot analysis and real time RT-PCR showed that the transcript expression level of Le.nik1 increases from mycelium to mature fruiting body. This suggests that Le.nik1 plays an essential role in fruiting body development. In situ hybridization of different fruiting body stage demonstrated the transcript localization of Le.nik1 in the developing hymenophore and trama cells, which reveals Le.nik1's role in fruiting body development. Real time RT-PCR results suggest the relationship between Le.nik1 and dicarboximide fungicides and oxidant. Yeast two-hybrid studies of Le.nik1 response regulator yields two novel interacting protein and they may also be related to fruit body development as shown by real-time RT-PCR and in situ hybridization. / Le.MAPK gene was isolated and identified by RNA fingerprinting of differentially expressed genes. Le. MAPK shows high sequence identity to the MAP kinase in other fungus includes U. maydis, N. crassa and S. cerevisiae. Le.MAPK was found to be interacting with Le.DRMIP from the yeast two hybrid analyses. Le.DRMIP is a novel gene with a predicted N-terminal mitochondrial signal peptide, suggesting that their interactions relate to the mitochondrial signaling pathway. The expression profiles of these two genes reveal their importance in fruiting body initiation and development; the Le.DRMIP transcript is localized predominantly in the developing young fruit body and gills, which further signifies its role in cell differentiation during mushroom development. These results suggest a model in which Le.MAPK and Le.DRMIP regulate mitochondrial signal transduction during fruit body development in L. edodes. / Through the studies of Le. MAPK and Le.nik1 , this work enhances our knowledge of the role and functions of these signal transduction genes in mushroom development. These studies can also help us to investigate the biological function of these signal transduction genes in fungi and other organisms. / Szeto Ying Ying. / "October 2006." / Adviser: Hoi Shan Kwan. / Source: Dissertation Abstracts International, Volume: 68-09, Section: B, page: 5750. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (p. 126-139). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

Chum Wing Yan Winnie. / "August 2006." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (p. 190-223). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese.

Xing, Lei. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 111-120). / Abstracts in English and Chinese. / ABSTRACT OF THESIS ENTITLED: --- p.I / 論文摘要 --- p.Ill / ACKNOWLEDGEMENTS --- p.IV / ABBREVIATIONS --- p.V / CONTENTS --- p.VI / LIST OF FIGURES --- p.X / LIST OF TABLES --- p.XII / Chapter CHAPTER 1: --- LITERATURE REVIEW --- p.1 / Chapter 1.1 --- Introduction --- p.1 / Chapter 1.1.1 --- Pectic substances --- p.1 / Chapter 1.1.2 --- Structure and classification of pectins --- p.2 / Chapter 1.1.3 --- Classification of pectinases --- p.4 / Chapter 1.1.4 --- Application of pectinases --- p.5 / Chapter 1.1.5 --- Production of pectinases --- p.5 / Chapter 1.2 --- Lentinula edodes as a source of pectinolytic enzymes --- p.12 / Chapter 1.2.1 --- Taxonomy and Life cycle of L. edodes --- p.12 / Chapter 1.2.2 --- Pectin-degrading enzymes in L edodes --- p.13 / Chapter 1.3 --- Expression systems for fungal pectinolytic enzymes --- p.16 / Chapter 1.4 --- Gene expression analysis --- p.19 / Chapter 1.5 --- Objectives and Long-term significance --- p.20 / Chapter CHAPTER 2: P --- EGTINASES IN L. EDODES --- p.23 / Chapter 2.1 --- Introduction --- p.23 / Chapter 2.2 --- Materials and Methods --- p.25 / Chapter 2.2.1 --- Fungal strains and growth conditions --- p.25 / Chapter 2.3.2 --- Gene models --- p.25 / Chapter 2.2.4 --- Enzyme activity assays --- p.26 / Chapter 2.3 --- Results --- p.30 / Chapter 2.3.1 --- Alignment of 24 candidate pectin-degradation gene models --- p.30 / Chapter 2.3.2 --- Conserved domains in protein sequences of 24 gene models --- p.30 / Chapter 2.3.3 --- Signal Peptide prediction of pectin-degradation gene models --- p.30 / Chapter 2.3.4 --- Pectinases activities in L. edodes --- p.31 / Chapter 2.3.5 --- Growth of mycelia of L. edodes on pectin and non-pectin media --- p.31 / Chapter 2.4 --- Discussion --- p.48 / Chapter 2.4.1 --- Elimination of non-pectinolytic genes --- p.48 / Chapter 2.4.2 --- Conserved domains and active sites of 6 polygalacturonases --- p.49 / Chapter 2.4.3 --- Pectinases activities in L. edodes --- p.49 / Chapter 2.4.4 --- Effect of pectin on the growth of mycelia in L. edodes --- p.50 / Chapter 2.5 --- Conclusion --- p.51 / Chapter CHAPTER 3: --- TRANSCRIPTIONAL PROFILING OF PECTINASES GENES IN L. EDODES --- p.52 / Chapter 3.1 --- Introduction --- p.52 / Chapter 3.2 --- Materials and Methods --- p.57 / Chapter 3.2.2 --- Strain cultivation --- p.57 / Chapter 3.2.3 --- RNA extraction and first strand cDNA synthesis --- p.58 / Chapter 3.2.4 --- Quantitative RT-PCR --- p.58 / Chapter 3.2.5 --- Data analysis --- p.59 / Chapter 3.3 --- Results --- p.62 / Chapter 3.3.2 --- RNA quality of various samples in L. edodes --- p.62 / Chapter 3.3.3 --- Transcription of 14 putative pectinases genes --- p.62 / Chapter 3.3.4 --- Transcription profiling of pectinases genes during the development of L. edodes --- p.62 / Chapter 3.3.5 --- Transcriptional levels of pectinases genes in mycelia of L. edodes grown in different media --- p.63 / Chapter 3.4 --- Discussions --- p.73 / Chapter 3.4.2 --- Transcription profiling of pectinases genes in L. edodes during four developmental stages --- p.73 / Chapter 3.4.3 --- Differential transcriptional levels of pectinases genes in L. edodes mycelia grown in two media --- p.73 / Chapter 3.4.4 --- Effect of pectic substrates on the pectinases genes transcription in mycelia of L. edodes --- p.74 / Chapter 3.5 --- Conclusion --- p.76 / Chapter CHAPTER 4: --- CLONING OF PECTINASES GENES AND THEIR HETEROLOGOUS EXPRESSION IN PICHIA PASTORIS --- p.77 / Chapter 4.1 --- Introduction --- p.77 / Chapter 4.2 --- Materials and methods --- p.79 / Chapter 4.2.2 --- Strain cultivation --- p.79 / Chapter 4.2.3 --- RNA extraction and first strand cDNA synthesis --- p.79 / Chapter 4.2.4 --- Cloning and sequencing of pectinases genes --- p.80 / Chapter 4.2.5 --- Subcloning and expression vector construction --- p.80 / Chapter 4.2.6 --- Growth of Pichia pastoris strains --- p.81 / Chapter 4.2.7 --- Transformation into P. pastoris and vivo screening of multiple inserts --- p.81 / Chapter 4.2.8 --- Expression of recombinant P. pastoris strains --- p.82 / Chapter 4.2.9 --- RNA extraction and transcription analysis of pectinases genes in recombinant Pichia strains --- p.83 / Chapter 4.2.10 --- Enzyme activity assays --- p.83 / Chapter 4.2.11 --- SDS-PAGE --- p.84 / Chapter 4.3 --- Results --- p.88 / Chapter 4.3.2 --- RT-PCR for full-length cDNA of 13 pectinases genes --- p.88 / Chapter 4.3.3 --- Cloning and sequences analysis of 4 putative pectinases genes --- p.88 / Chapter 4.3.4 --- Construction of expression vectors of pectinases genes and transformation to P. pastoris --- p.88 / Chapter 4.3.5 --- Screening of multiple inserts clones --- p.88 / Chapter 4.3.6 --- Recombination and integration of pectinases genes in P. pastoris --- p.89 / Chapter 4.3.7 --- Transcription and expression of pectinases genes in recombinant Pichia strains. --- p.89 / Chapter 4.4 --- Discussion --- p.104 / Chapter 4.4.2 --- cDNA sequences of 4 pectinases genes --- p.104 / Chapter 4.4.3 --- Heterologous expression of 2 pectinases genes in P. pastoris --- p.104 / Chapter 4.4.4 --- Characterization of the pectinases expressed by recombinant Pichia strains --- p.106 / Chapter 4.5 --- Conclusion --- p.108 / Chapter CHAPTER 5: --- CONCLUDING REMARKS --- p.109 / REFERENCES --- p.121

Luo, Xiao. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 118-134). / Abstracts in English and Chinese. / Abstract --- p.i / 摘要 --- p.iii / Acknowledgements --- p.iv / Abbreviations --- p.v / Contents --- p.vi / List of Figures --- p.ix / List of Tables --- p.xii / Chapter Chapter 1 : --- Literature Review --- p.1 / Chapter 1.1 --- Lentinula edodes --- p.1 / Chapter 1.1.1 --- Introduction and taxonomy --- p.1 / Chapter 1.1.2 --- Nutritional values and medical values --- p.2 / Chapter 1.2 --- Life cycle and morphology --- p.5 / Chapter 1.3 --- Lignocellulolytic system in wood-rotting fungi --- p.9 / Chapter 1.3.1 --- Structures of lignin --- p.9 / Chapter 1.3.2 --- Wood-rotting fungi --- p.11 / Chapter 1.3.3 --- Lignin degradation by white rot fungi --- p.12 / Chapter 1.3.4 --- Ligninolytic enzymes --- p.16 / Chapter 1.3.4.1 --- Lignin peroxidase --- p.16 / Chapter 1.3.4.2 --- Maganese peroxide --- p.16 / Chapter 1.3.4.3 --- Laccases --- p.19 / Chapter 1.3.5 --- Potential Industrial application of liglinolytic enzymes --- p.22 / Chapter 1.3.6 --- Ligninolytic enzymes in L. edodes --- p.23 / Chapter 1.4 --- Expression systems for fungal ligninolytic enzymes --- p.24 / Chapter 1.5 --- Aim of this project --- p.27 / Chapter 1.6 --- Long-term significance --- p.28 / Chapter Chapter 2: --- Sequence analysis of ligninolytic enzymes from Lentinula edodes --- p.29 / Chapter 2.1 --- Introduction --- p.29 / Chapter 2.2 --- Materials and methods --- p.32 / Chapter 2.2.1 --- Phylogenetic study and signal peptide prediction of the decuced ligninolytic enzymes --- p.32 / Chapter 2.2.2 --- Comparison ligninolytic enzymes of L. edodes and other basidiomycetes fungi --- p.32 / Chapter 2.3 --- Results --- p.34 / Chapter 2.3.1 --- Protein sequence analysis and signature sequences identification of L. edodes laccases --- p.34 / Chapter 2.3.2 --- Protein sequence analysis of L. edodes manganese peroxidases --- p.34 / Chapter 2.3.3 --- Phylogenetic study of ligninolytic genes from L.edodes --- p.35 / Chapter 2.4 --- Disscussion --- p.52 / Chapter Chapter 3: --- Transcription profiling of ligninolytic enzymes from Lentinula edodes --- p.56 / Chapter 3.1 --- Introduction --- p.56 / Chapter 3.2 --- Materials and Methods --- p.61 / Chapter 3.2.1 --- Strain cultivation --- p.61 / Chapter 3.2.2 --- "RNA extraction, mRNA isolation and cDNA synthesis" --- p.63 / Chapter 3.2.3 --- RNA Quality Estimation --- p.64 / Chapter 3.2.4 --- cDNA synthesis --- p.65 / Chapter 3.2.5 --- Primer verification --- p.66 / Chapter 3.2.6 --- Quantitative RT-PCR --- p.66 / Chapter 3.3 --- Results --- p.70 / Chapter 3.3.1 --- RNA quality estimation --- p.70 / Chapter 3.3.2 --- Quantification real time PCR --- p.70 / Chapter 3.3.3 --- Transcriptional profiling of laccases during the development of L edodes --- p.70 / Chapter 3.3.4 --- Transcriptional profiling of MnPs during the development of L edodes --- p.71 / Chapter 3.3.5 --- Transcript level analysis of laccases from in mycelia grown on lignocelluloses medium and non -lignocelluloses medium --- p.71 / Chapter 3.3.6 --- Transcript level analysis of MnPs in mycelia grown on lignocelluloses medium and non -lignocelluloses medium --- p.72 / Chapter 3.3.7 --- Differential expression of laccases from L. edodes grownin lignocelluloses medium during mycelia stage --- p.72 / Chapter 3.3.8 --- Differential expression of laccases from L. edodes grownin lignocelluloses medium during mycelia stage --- p.72 / Chapter 3.4 --- Discussion --- p.87 / Chapter 3.4.1 --- Transcriptional profiling of laccases and MnPs during four developmental stages --- p.87 / Chapter 3.4.2 --- Transcriptional profiling of laccases and MnPs in mycelium grown in lignocelluloses and non-lignocelluloses medium --- p.88 / Chapter 3.4.3 --- Temporal differential expression of laccases and manganese peroxidases --- p.90 / Chapter 3.5 --- Conclusion --- p.92 / Chapter Chapter 4: --- "Cloning and heterologous expression of Lentinula edodes laccase, lac1B, in yeast Pichia pastoris" --- p.93 / Chapter 4.1 --- Introduction --- p.93 / Chapter 4.2 --- Materials and Methods --- p.95 / Chapter 4.2.1 --- Strain cultivation --- p.95 / Chapter 4.2.2 --- First strand cDNA synthesis --- p.95 / Chapter 4.2.3 --- Construction of cDNA library --- p.95 / Chapter 4.2.4 --- Signal peptide prediction of Iac1 B --- p.96 / Chapter 4.2.5 --- Cloning of native laccase into Pichia pastoris expression vector --- p.96 / Chapter 4.2.6 --- Screening for positive colonies --- p.97 / Chapter 4.2.7 --- Construction of pool of recombinant vector --- p.97 / Chapter 4.2.8 --- Transformation of P. pastoris --- p.98 / Chapter 4.2.9 --- Screening for expression cassette into Pichia pastoris --- p.98 / Chapter 4.2.10 --- Enzyme Activity assay --- p.99 / Chapter 4.2.11 --- SDS-PAGE --- p.100 / Chapter 4.3 --- Results --- p.103 / Chapter 4.3.1 --- Screening for positive colonies with recombinant vector in TOP10 --- p.103 / Chapter 4.3.2 --- Screening for expression cassette from transform ants of P pastoris --- p.103 / Chapter 4.3.3 --- Enzyme activity assay --- p.103 / Chapter 4.3.4 --- SDS-PAGE --- p.104 / Chapter 4.4 --- Disscussion --- p.109 / Chapter Chapter 5: --- Concluding Remarks --- p.111 / Reference --- p.118

Submitted by Nathália Faria da Silva (nathaliafsilva.ufv@gmail.com) on 2017-06-13T14:33:59Z No. of bitstreams: 1 resumo.pdf: 18903 bytes, checksum: 54fcdb88877bc035ef66ceb254f308c8 (MD5) / Made available in DSpace on 2017-06-13T14:33:59Z (GMT). No. of bitstreams: 1 resumo.pdf: 18903 bytes, checksum: 54fcdb88877bc035ef66ceb254f308c8 (MD5) Previous issue date: 2003-06-13 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / A adequação das etapas de processamento mínimo e as alterações nas características químicas e na microbiota contaminante de cogumelos shiitake minimamente processados e estocados a 7, 10 e 15oC, em embalagem de poliestireno expandido foram avaliadas. Verificou-se que a centrifugação de porções de 250 g do cogumelo por períodos superiores a 2 minutos resultou em danos físicos e murchamento no chapéu do shiitake. A sanitização dos cogumelos em solução contendo 200 ppm de cloro livre, por 10 minutos, à temperatura ambiente, reduziu, significativamente, 1,93 ciclos logaritmo na população de aeróbios mesófilos. A sanitização com 1% de ácido acético e 1% de ácido lático reduziu 1,3 ciclos logaritmos desta microbiota. O ácido acético foi efetivo para reduzir 3,73 ciclos logaritmos na população de Pseudomonas, enquanto os outros sanitizantes não apresentaram redução significativa em relação ao controle, onde se usou apenas água. A imersão das amostras de cogumelos nas soluções de clorado orgânico, ácido acético e lático não promoveu redução significativa na população de psicrotróficos aeróbios e de bolores e leveduras. Constatou-se que a sanificação com ácidos orgânicos promoveu o escurecimento pronunciado dos cogumelos que apresentaram valores mínimos de L* de 27, enquanto aqueles não sanitizados ou sanitizados com clorado orgânico os valores mínimos de L* variaram em torno de 32, no 15 o dia de estocagem. Constatou-se uma redução acentuada na concentração de oxigênio dentro das embalagens de cogumelos shiitake minimamente processados, no início do tempo de estocagem a 7, 10 e 15oC. Essa concentração de O 2 manteve-se baixa durante 15 dias de estocagem e não diferiu, significativamente, nas diferentes temperaturas avaliadas. O valor inicial do pH shiitake minimamente processado foi de aproximadamente 5,6 e decresceu nos cinco primeiros dias de estocagem. Os cogumelos shiitake minimamente processados escureceram ao longo da estocagem e, esse escurecimento foi mais acentuado quanto maior a temperatura de armazenamento. A população de mesófilos, psicrotróficos aeróbios e fungos predominaram em relação aos psicrotróficos anaeróbios, Pseudomonas e coliformes em todas as amostras analisadas e aumentou, o equivalente a 3 e 5,7 ciclos logaritmos, ao longo da estocagem a 7, 10 e 15oC. A caracterização morfo- tintorial de isolados psicrotróficos evidenciou a presença de bastonetes, Gram- negativos e a identificação bioquímica revelou tratar-se de espécies do gênero Pseudomonas. Os resultados do teste de aceitação permitiram estimar que os cogumelos shiitake minimamente processados mantiveram aparência aceitável, no 10o dia a 7oC, por um período inferior a 5 dias a 10oC e, aproximadamente, 3 dias a 15oC. A atividade de polifenol oxidase nos cogumelos processados foi, em geral, maior nos produtos resfriados a 15oC, em relação àqueles armazenados a 7 e 10oC. O uso de antioxidantes, como ácido cítrico e ácido ascórbico reduziu a atividade de PPO dos cogumelos shiitake minimamente processado, resultando em menor escurecimento da superfície do chapéu. Os cogumelos tratados com ácido cítrico apresentaram menor atividade de PPO e maiores valores de L*, ou sejam, tornaram-se menos escuros em razão da uma menor atividade da enzima. / The adaptation of minimal processing stages and alterations in the chemical characteristics and contaminating microbiota of minimally processed shiitake mushrooms, stored at 7, 10 and 15 o C in expanded polyethylene trays were evaluated. It was verified that centrifugation of 250 g portions of mushroom for periods over 2 min caused physical damage and wilting to the shiitake cap. Sanitation of the mushrooms in solution containing 200 ppm of chlorine at room temperature, for 10 min, significantly reduced 1.93 logarithm cycles in the population of mesophyll aerobic. Using 1% acetic acid and 1% lactic acid reduced 1.3 logarithm cycles of this microbiota. Acetic acid was effective in reducing 3.73 logarithm cycles in the Pseudomonas population, while the other sanitation solutions did not present a significant reduction compared to the control using only water. The immersion of the mushroom in organic chloride, acetic and lactic acid did not promote a significant reduction in the population of psychotropic aerobic and fungi. Sanitation using organic acids promoted a marked darkening of the mushrooms, which presented minimal L* values of 27, while non-sanitized ones or those sanitized with organic chloride solution had minimal L* values ranging around 32, on the 15 th storage day. There was a marked reduction in oxygen concentration inside the containers of minimally processed shiitake mushrooms, at the beginning of the storage time at 7, 10, and 15 o C. This O 2 concentration ixremained low during 15 storage days and did not differ significantly under the different temperatures evaluated. The initial pH value of the minimally processed shiitake was approximately 5.6, decreasing in the first five days of storage. The minimally processed shiitake mushrooms became dark along storage with this darkening becoming more accentuated as the storage temperature increased. The mesophilic, psychrotrophic aerobic and fungi populations predominated compared to the anaerobic psychrotrophic, Pseudomonas, and coliforms in all the samples analyzed, increasing on an average 3, 4 and 5.7 logarithm cycles throughout storage at 7, 10 and 15 o C. The morpho-tinctorial characterization of psychrotrophic isolates indicated the presence of Gram-negative bacillus and the biochemical identification showed they belonged to the Pseudomonas genus. The results of the acceptance tests allowed estimating that minimally processed shiitake mushrooms kept an acceptable appearance on the 10 th day at 7 o C for a period shorter than 5 days at 10 o C, and approximately, 3 days at 15 o C. The polyphenol oxidase (PPO) activity in the processed mushrooms was in general, higher than in the products cooled at 15 o C, compared to those stored at 7 and 10 o C. The use of antioxidants, such as citric acid and ascorbic acid, reduced the PPO activity in the minimally processed shiitake mushrooms, resulting in less darkening of the mushroom surface cap. The citric acid-treated mushrooms presented lower PPO activity and higher L* values, i .e., they became less dark due to a lower enzyme activity.

Submitted by Reginaldo Soares de Freitas (reginaldo.freitas@ufv.br) on 2016-11-04T15:52:41Z No. of bitstreams: 1 texto completo.pdf: 323897 bytes, checksum: d13466701013a134479a1272fefcee34 (MD5) / Made available in DSpace on 2016-11-04T15:52:41Z (GMT). No. of bitstreams: 1 texto completo.pdf: 323897 bytes, checksum: d13466701013a134479a1272fefcee34 (MD5) Previous issue date: 2004-11-11 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / O objetivo deste estudo foi avaliar pré-tratamentos e processos de congelamento para a conservação de cogumelo Lentinula edodes shiitake . Características físico-químicas e microbiológicas de cogumelos obtidos de produtores da região de Viçosa, colhidos, transportados em caixas com gelo e processados no mesmo dia foram avaliadas a cada trinta dias durante o período de armazenamento de quatro meses. As etapas do processamento foram: seleção, lavagem em água corrente, sanitização com produto organoclorado, branqueamento ou adição de antioxidante, centrifugação para eliminar o excesso de água, seguidas de embalagem e congelamento lento em congelador doméstico ("freezer") ou congelamento rápido com Nitrogênio líquido e embalagem . As amostras congeladas foram armazenadas em congelador doméstico ("freezer") a -18oC. O branqueamento não foi satisfatório porque provocou perda de água excessiva e perda de textura, resultando em cogumelos com aparência indesejável. O tratamento com composto organoclorado e a adição de antioxidantes foram mais efetivos na manutenção da qualidade dos cogumelos durante o armazenamento, com maior redução da microbiota inicial, apresentando após o descongelamento, valores de textura e pH mais próximos aos dos cogumelos frescos. Sem o branqueamento, a atividade das Polifenol Oxidases ( PPOs ) das amostras congeladas aumentou ao longo do tempo causando escurecimento dos cogumelos e redução dos valores de L* porém as amostras congeladas com nitrogênio líquido apresentaram, durante todo o período de armazenamento, menor perda de vitamina D e de água, melhor textura e aparência que aquelas congeladas pelo método lento. Até o primeiro mês de armazenamento, para as amostras congeladas pelo processo tradicional e até o segundo mês, para as amostras congeladas pelo processo rápido, as características organolépticas dos cogumelos depois de descongelados foram consideradas satisfatórias, mas após esses períodos os cogumelos começaram a apresentar escurecimento, mau cheiro e perda de textura inaceitáveis. / This experiment was conducted to evaluate pre-processing and freezing conditions for the preservation of Lentinula edodes mushrooms ("Shiitake"). Mushrooms cultivated in areas near by Viçosa-MG were collected, kept in ice-boxes during transportation and processed in the same day. Frozen samples stored for four months in domestic freezers ( -18 oC ) were evaluated with 30 days intervals. Pre-processing included selection, washing in running water, sanitization with an organic chlorinated product, blanching or antioxidant addition and centrifugation to eliminate excess water, followed by packaging and "conventional freezing" in a domestic freezer or "Individual quick freezing" (IQF) with liquid Nitrogen followed by packaging. Blanching was not satisfactory because of excessive water and textures losses producing mushrooms with bad appearance. Treatment with chlorinated product and antioxidant adition were more effective for keeping the quality of frozen samples, with lower microbial counts and, after thawing, texture, color and pH closer to those of fresh mushrooms. Without blanching, activity of PPO increased during storage causing darkening of the frozen mushrooms and reducing L* values. Samples frozen by IFQ presented smaller losses of Vitamin D and water and better texture and appearance than those processed by conventional freezing. After thawing the quality of the mushrooms frozen by the slow process was better than acceptable for 30 days while the quality of the mushrooms frozen by IFQ was better than acceptable for 60 days (IFQ better, always). After those periods, the frozen samples started to show excessive losses texture, darkening, off- flavor and chiefly obnoxious smell.

Made available in DSpace on 2014-06-11T19:31:34Z (GMT). No. of bitstreams: 0 Previous issue date: 2007-05-24Bitstream added on 2014-06-13T20:41:59Z : No. of bitstreams: 1 andrade_mcn_dr_botfca.pdf: 1132419 bytes, checksum: 28616e10ef21df6298a36b3b132aeeb9 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Mycelium growth, production and bromatologicals characteristics of shiitake in function of lineages and chemical and physical properties of eucalyptus clones and species were evaluated. In Experiment 1, mycelium growth of two Lentinula edodes (Berk.) Pegler (LE-95/01 and LE-96/18) species in culture mediums prepared with sawdust extract from seven species (E. saligna, E. grandis, E. urophylla, E. camaldulensis, E. citriodora, E. paniculata e E. pellita) and three eucalyptus clones (hybrid E. grandis x E. urophylla) was analyzed. The experimental design was totally randomized, in 2x10 factorial design, totalizing 20 treatments with 10 repetitions, being that each repetition corresponded to one Petri dish. In Experiment 2, mycelium growth of eight L. edodes lineages (LE-96/17, LE-95/02, LE-95/07, LE-98/55, LE-96/18, LE-95/01, LE-96/13 and LE-98/47) in culture mediums prepared with sawdust extract from Eucalyptus spp was evaluated. The experimental design was totally randomized, with 8 treatments and 8 repetitions, being that each repetition corresponded to one Petri dish. In Experiment 3, production and bromatological characterization of two Lentinula edodes lineages cultivated in seven species and three clones of eucalyptus was evaluated. The experimental design was totally randomized, in 2x10 factorial design, totalizing 20 treatments with 40 repetitions, being that each repetition corresponded to one log. In Experiment 4, physical and chemical properties of seven species and three clones of eucalyptus before and after the cultivation of two L. edodes lineages was evaluated. The experimental design was totally randomized, in 2x10 factorial design, totalizing 20 treatments with 9 repetitions, being that each repetition corresponded to one log. The culture medium that provided highest averages of mycelium growth of L. edodes lineages LE-95/01 and LE-96/18 was the one with... (Complete abstract click electronic access below)

In this study, a novel compound was isolated and purified from the solid culture medium (potato dextrose agar) of shiitake 1358 strain through series of methods, such as ethanol precipitation, macroporous resin column separation, semi-preparative high performance liquid chromatography separation and preparative thin-layer chromatography separation. Analyzing spectra from fourier transform infra-red spectroscopy, gas chromatography-mass spectrometry, 1-dimension and 2-dimension nuclear magnetic resonance, the chemical structure of the novel compound was determined and named as 4-amino-5,6-dihydrobenzo[d]oxonine-2,7(1H,4H)-dione. It could inhibit the proliferation of HL-60 leukemia cells significantly and with an IC50 of 1.56 mug/ml (7.123 mumol/L) in the 72-hour treatment. From the results, it is suggested that this compound could activate the G2 phase checkpoint control of the cell cycle to arrest the cell cycle in G2 phase. In addition, it could suppress the replicative DNA synthesis to inhibit the proliferation of HL-60 leukemia cells. The more important is that this compound can induce the apoptosis of HL-60 leukemia cells significantly through intrinsic and extrinsic apoptotic pathways. The compound could induce intrinsic and extrinsic apoptosis through the regulation of the apoptosis-related proteins, such as Fas ligand, Bax, Bcl-2, Caspase 8, Caspase 9, and Caspase 3. For intrinsic pathway, the compound might upregulate Bax, downregulated Bcl-2, activated the Caspase 9, subsequently activated Capase 3, and ultimately led to cell death. For extrinsic pathway, the compound upregulated the Fas ligand, cleaved and activated Procaspase 8 to active Caspase 8, further cleaved and activated Procaspase 3 to active Caspase 3 to commit the cells to apoptosis. / Leukemia is a malignant cancer that involves the bone marrow and blood circulation systems. Leukemia results in the uncontrolled growth of abnormal (leukemic) white blood cells and may also invade other organs, including the liver, spleen, lymph nodes, testes, and brain. In 2007, about 44,240 new cases of leukemia were diagnosed and 21,790 patients died from all types of leukemias in USA. / Shiitake was first cultivated in China more than 800 years ago. It is the second most commonly cultivated edible mushrooms in the world nowadays. For a long time, shiitake has been valued for its unique taste and flavor and as a medicinal invigorant. According to ancient Chinese medicinal theory, consumption of shiitake was in favor of long life and good health. In China and Japan, shiitake has been used as both a food and a medicinal herb for thousands of years. It is the source of several well-studied preparations with proven pharmacological properties, especially the polysaccharide lentinan. Currently, most researches concentrate on the anticancer activities of the extracts from the fruiting body of shiitake, especially polysaccharides. Report about the anti-cancer effects of other components from the shiitake mushroom is scarce. The objectives of this investigations were: (1) to study the anticancer activities of brownish substances obtained during the solid medium culture of shiitake on specific cancer cell unes, especially HL60 cancer cell line; (2) to isolate and characterize the active compound(s) in the brown mushroom exudates; and (3) to propose the possible mechanism of actions, especially the function of the bcl-2 family genes and proteins. / by Guo, Yuming. / Adviser: Chung Hale Yin. / Source: Dissertation Abstracts International, Volume: 70-06, Section: B, page: 3314. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 188-199). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.