Academic literature on the topic 'Submerged cultivation'

Wheat chaff as an agricultural waste represents a cheap raw material for biotechnological processes. With its lignocellulosic composition, it is suitable for producing hydrolytic enzymes for second generation renewable fuel production technologies. The aim of this work was to optimize the process parameters (cultivation temperature 25?35?C, pH value 4?6 and cultivation time 3?7 days) of the cultivating fungi (Trichoderma reesei QM 9414) on a media based on wheat chaff by submerged and solid-state techniques, in order to enhance and compare the two types of simultaneous cellulase and xylanase production. Optimal conditions for the submerged fermentation were 29.65?C for temperature, pH 4.27 and 7 days of cultivation, while for the solid-state fermentation, the optimal conditions were 28.01?C, pH 6.00 and 7 days. The cellulolytic and xylanolytic activities of the obtained cultivation broth filtrates were 0.0535 and 0.1676 U mL-1 for the submerged fermentation, and 0.0407 and 0.1401 U mL-1 for the solid-state fermentation, respectively, and with a 26.77 and 13.39 % enhancement of enzyme activity for submerged fermentation, and a 22.96 and 42.66 % enhancement for solid-state fermentation, respectively, compared to the results obtained before optimization.

This paper deals with the biosynthetic capacity for ochratoxin A (OTA) production by Aspergillus ochraceus E'G isolate derived from A. ochraceus CBS 108.08 strain, during 2007. Preliminary analysis of fungal potential for the production of OTA were performed according to the modified method of Filtenborg et al. (1983). Toxin production was tested in the following liquid media: (i) glucose-peptone-yeast extract broth (GPY - pH 5.6), (ii) potato-dextrose broth (PDB - pH 6.9), (iii) yeast extract-sucrose broth (YES - pH 6.5), and (iv) YES broth supplemented with 0.23 mg/l ZnSO4 x 5 H2O (YESZn - pH 6.5) after stationary and submerged cultivation. Dynamics of OTA biosynthesis was tested after the cultivation of A. ochraceus E'G on natural solid substrates, such as wet sterilized rice, corn and wheat grain. Cultivations were performed during different time periods (ranging from four days to few weeks) at different temperatures (ranging from 21?C to 30?C). The presence of OTA was determined as follows: (i) in liquid media according to the method of Balzer et al. (1978) modified by Bocarov-Stancic et al. (2003), and (ii) in the solid substrates according to the Serbian official methods for sampling and analyzing of fodder (Official Gazette of SFRY, No. 15/87). After the cultivation of A. ochraceus E'G isolate in liquid media, the highest yield of OTA (6.4 mg/l) was obtained after submerged cultivation in PDB (4 days, 128 rpm, 21-23?C). In the case of cultivation on solid substrates, the highest amount of OTA (800.0 mg/kg of dry matter) was recorded after several week long cultivation on wheat grain at 30?1?C.

Ganoderma applanatum belongs to the group of white-rot fungi, due to a well-developed ligninolytic enzyme system. White-rot fungi have attracted great scientific attention in recent years, especially with respect to their enzymatic potential for the bioremediation of persistent pollutants. Contrary to G. lucidum, which medicinal properties, as well as ligninolytic enzyme system have been extensively studied, enzymatic system of G. applanatum has not been studied yet. Thus, the aim of this study was to analyze the dynamics of laccase, Mn-dependent peroxidase, and versatile peroxidase activity during submerged and solid state cultivation on two selected plant raw materials. Enzyme activity was determined spectrophotometrically after 7, 10 and 14 days of cultivation. The peak of laccase activity (220.14 Ul-1) was noted after 14 days of submerged wheat straw fermentation. Maximum level of Mn-dependent peroxidase (110.91 Ul-1) and versatile peroxidase (116.20 Ul-1) activity was obtained in the medium with oak sawdust after 14 days of submerged cultivation.

This study aims to (1) examine the physical and chemical aspects of marine oceanography, (2) determine the suitability of location and swimming crab culture techniques in submerged net cages, (3) find out the management strategies of crab culture management in submerged net cages. The results showed that the suitability of waters for crab cultivation activities in the karamba nets was submerged by using indicators of temperature, salinity, oxygen, pH and brightness obtained two suitability criteria, namely sesai and not. The results of the suitability analysis of crab cultivation land using a Geographic Information System (GIS) obtained two criteria for suitability of cultivated land with an area of each appropriate category that is 46,785.32 ha and not according to 1,152.4 ha. The results of management strategies that can be applied to realize integrated management of small crab culture include: 1) Development and socialization of small crab culture; 2) Management of integrated small crab cultivation; 3) Law enforcement related to the destruction of coastal ecosystems 4) Prohibition of capturing crabs laying eggs; 5) Construction of small crab hatcheries for small crab cultivation needs; 6) Development and provision of venture capital.

Chitin-glucan complex is a fungal origin copolymer that finds application in medicine and cosmetics. Traditionally, the mycelium of Micromycetes is considered as an industrial chitin-glucan complex source. Basidiomycete Schizophyllum commune submerged cultivation for chitin-glucan complex production was studied. In different S. commune strains chitin-glucan complex composed 15.2 +/- 0.4 to 30.2 +/- 0.2% of mycelium dry weight. Optimized conditions for chitin-glucan complex production (nutrient medium composition in g/l: sucrose - 35, yeast extract - 4, Na2HPO4*12H2O - 2.5, MgSO4*7 H2O - 0.5; medium initial pH 6.5; aeration intensity 21 of air per 11 of medium; 144 hours of cultivation) resulted in 3.5 +/- 0.3 g/l complex yield. Redirection of fungal metabolism from exopolysaccharide synthesis to chitin-glucan complex accumulation was achieved most efficiently by aeration intensity increase. Chitin-glucan complex from S. commune had the structure of microfibers with diameter 1-2 microm, had water-swelling capacity of 18 g/g, and was composed of 16.63% chitin and 83.37% glucan with a degree of chitin deacetylation of 26.9%. S. commune submerged cultivation is a potent alternative to Micromycetes for industrial-scale chitin-glucan complex production.

A goma xantana continua sendo o polissacarídeo microbiano mais produzido no mundo. Características como aumento da viscosidade em soluções, agente de geleificação, estabilidade frente aos diversos tratamentos despertam um interesse especial para a indústria de alimentos, porém, a aplicação para fins não alimentícios vem crescendo significativamente, como é o caso da indústria petrolífera e têxtil. A utilização de substratos alternativos e baratos para a produção de biomoléculas de interesse comercial, como resíduos agroindústrias, vem sendo alvo de virias pesquisas na atualidade. Sendo assim, neste trabalho, produzimos goma xantana utilizando a casca de soja como substrato para a Xanthomonas campestris. Utilizou-se um planejamento experimental e da metodologia de superfície de resposta a fim de identificar as melhores condições de produção de goma xantana em cultivo semi-sólido em biorreatores estáticos (CSSE). As melhores condições para este cultivo foram: temperatura de 31.2ºC, aeração de 467.5 L.min-1 e densidade óptica do inóculo de 0,929 em 600 nm. Em paralelo, conduziu-se um estudo da produção de goma xantana em cultivo submerso (CSm) e em cultivo semi-sólido agitado (CSSA), fazendo-se um comparativo entre os três sistemas quanto à produção e a viscosidade do exopolissacarídeo. Quanto à conversão de casca de soja em goma, o CSSA foi o que mais converteu, chegando a 19%, seguido de 10% no CS e 8% no SSE. A viscosidade da goma em solução chegou a 1550 cP na taxa de cisalhamento de 1 s-1 para SSA. A utilização da casca de soja como substrato e suporte de crescimento microbiano, mostrou-se adequado nessas condições.
The xanthan gum is still the most produced microbial polysaccharide in the world. Characteristics as the increase of viscosity in solutions, geleificant agent, stability in several treatments, bnng a special interest for the food industry, however, the application for non-food applications has been increasing significantly, as the case of the textile and petroleum industry. The utilization of an alternative and non-expensive substrate for the biomolecules production of commercial interest, as agro-industry residues, has been the aim of some researches nowadays. Thus, in this work, we produce xanthan gum using soybean hull as substrate for the Xanthomonas campestris. An experimental factorial design and response surface methodology was used in order to identify the best conditions of production of xanthan gum in solid-state cultivation in static bioreactors (SSSC). The best conditions for this cultivation were temperature of 3 1.2ºC, aeration of 467.5 L.min-1 and optic density of inoculum of 0,929 in 600 nm. At the same time, a study of the production of xanthan gum in submerged cultivation (SmC) and solid-state agitated cultivation (ASSC) has been conducted, comparing the three systems in relation to the production and the viscosity of the exopolysaccharide. In relation to the conversion of the soybean hull into gum, the SSA was the one with the highest rate of conversion, reaching 19%, followed by 10% in CS and 8% in the SSS. The viscosity of the gurn in solution reached 1550 cP in the shear rate of 1 s-1 for SSA. The use of the soybean hull as substrate and support of microbial growth has shown adequate in these conditions.

Chitosan-glucan complex is fungal origin copolymer that finds application in medicine and cosmetics. Traditionally mycelium of Aspergillus and Penicillium is considered as industrial chitosan-glucan complex source, though utilization of Micromycetes in biotechnological productions is sometimes undesirable. The aim of the work was to study the possibility of Basidiomycete Schizophyllum commune submerged cultivation for industrial scale chitosan-glucan complex production use. Within the work there was studied effect of cultivation conditions (type and concentration of carbon sources in nutrient medium, ratio of carbon source to nitrogen source, medium initial pH and aeration intensity) on Sch. commune #127 mycelium growth, chitosan-glucan complex formation and exopolysaccharide synthesis. As the result, the method for chitosan-glucan complex production increase and exopolysaccharide synthesis suppression was suggested. Chitosan-glucan complex from Sch. commune #127 submerged mycelium was separated by successive alkali and acid treatments. Effects of alkali concentration and application technique, and type of acid on physical and chemical properties of chitosan-glucan complex were described. Analytical methods for in process control and final product characteristics were suggested.

Xanthan is a well-known extracellular polysaccharide, produced by a Gram negative bacterium Xanthomonas campestris (X. campestris) under aerobic conditions. Solutions of xanthan exhibit high viscosities and non-Newtonian behaviour even at low concentrations. This biopolymer has a wide range of valuable commercial and industrial applications, for example; it can be used as a food thickening agent and a stabilizer in some other industries. Traditionally the production of xanthan has predominantly been performed in stirred tank fermenter (STR). This study sought to compare the cultivation of the bacterium, X. campestris for the production of the viscous biopolymer xanthan gum in two different reactor systems, a novel oscillatory baffled reactor (OBR) and the conventional industry workhorse, the stirred tank reactor (STR). Overall biopolymer production occurred at similar rates in the well stirred and aerated STRs, albeit at the cost of higher energy inputs for mixing and aeration. Despite much previous literature promoting the use of the OBR for transporting and reacting very viscous systems, this was the first actual study attempting to investigate the use of the OBR for a highly viscous non-Newtonian fermentation process. The experimental results show that xanthan production was similar in the OBR than in the STR, the OBR is however readily suitable for the cultivation of xanthan. The probable reasons for the inability of the OBR to match the production rates of the STR may well lie in the complex nature of this fermentation process. Unlike a previous study on pullulan production (Gaidhani 2004) where the OBR outperformed the STR, X. campestris initially needs high oxygen transfer rates and the OBR, although it provides good bulk mixing and low energy consumption, seemed unable to equal the STR in this respect, especially in a very viscous system. The result shows that xanthan production in the OBR was similar to the equivalent process in the STR. In order to attempt to improve the OBR a number of technical modifications were made including a novel sparger design to improve gas dispersal. These were not successful in improving xanthan production. Similarly, attempts to achieve improvements via wider amplitude ranges led to damage to the equipment. The conclusion was that significant improvements to the physical robustness of the OBR were necessary before it could be successfully used to process highly viscous bio-fluids.

Proteases ácidas pertencem a um importante grupo de enzimas industriais produzidas por fungos filamentosos, com aplicações na indústria de alimentos, de couro, farmacêutica e de cosméticos. O objetivo principal deste trabalho foi avaliar a produção de proteases ácidas extracelulares de fungos filamentosos isolados do solo do cerrado do centro-oeste brasileiro. Inicialmente, foi realizada uma triagem para avaliar a capacidade de 17 linhagens de fungos quanto à produção de protease em meio de cultura contendo Agar-leite. O fungo Aspergillus foetidus foi selecionado como melhor produtor de protease ácida extracelular. Visando à otimização da produção de proteases pelo fungo selecionado, avaliou-se a influência de diversos fatores no cultivo (pH, temperatura, agitação e diferentes fontes de nitrogênio e carbono). Após essa etapa, um planejamento experimental estatístico foi realizado com as variáveis independentes temperatura, pH inicial do meio e fonte de carbono e nitrogênio. A produção máxima de protease foi encontrada (63,7 U/mL) nas condições: pH inicial do meio igual a 7,0 a 28 ºC, 150 rpm em peptona 2% (p/v). Os estudos em biorreator demonstraram produção de protease nas condições de agitação e aeração iguais à 300 rpm e 1,0 vvm, após 120 h de cultivo. Os ensaios com diferentes temperaturas para a estimativa dos parâmetros termodinâmicos demonstraram que a protease ácida produzida pelo fungo é altamente estável apresentando máxima atividade em pH 5,0 e temperatura ótima igual a 55ºC. E, finalmente, para a purificação da enzima foi realizada cromatografia de gel-filtração. A enzima apresentou massa molecular de 50,6 kDa, e a análise do zimograma confirmou a atividade proteolítica. Além disso, a protease purificada foi inibida pelo composto pepstatina, indicando uma característica de protease ácida. Esses resultados obtidos demonstram um fungo filamentoso produtor de uma nova protease ácida com potencial aplicação para indústria farmacêutica e de cosméticos.
The acid proteases belong to the most important group of industrial enzymes produced by filamentous fungi, with applications in the food, leather, pharmaceutical and cosmetics industries. This study aimed the evaluation of extracellular acid proteases production from filamentous fungi isolated from different samples of the midwestern Brazil cerrado. Initially, a screening was performed to assess the ability of the 17 strains of yeast for production of protease-agar medium containing milk culture. The Aspergillus foetidus was selected as the best producer. Aimed at optimizing the production of proteases by the selected fungus, first evaluated the influence of various factors on the cultivation (pH, temperatura, agitation and different sources of nitrogen and carbon). After this step, a statistical experimental design was carried out with the independent variables temperatura, initial pH of the medium and source of carbon and nitrogen. The best conditions for protease production were (63.7 U / mL): initial pH values greater than 7.0, at 28 °C, 150 rpm peptone 2% (w/v). Aiming future production of this protease in industrial scale, studies have shown better in bioreactor protease production under the conditions of agitation and aeration equal to 300 rpm and 1.0 vvm, after 120 h of cultive. The tests at different temperaturas to estimate the thermodynamic parameters showed that the acid protease produced by the fungus is highly stable with maximum activity at pH 5.0 and optimum temperatura of 55 °C. And finally, for the purification of the enzyme were performed gel-filtration chromatography. The enzyme had a molecular mass of 50.6 kDa, and the analysis of the zymogram showed a proteolytic band. Furthermore, the purified protease was inhibited by pepstatin compound, indicating a feature of acid protease. These results demonstrate a new filamentous fungus producing acid protease with potential application to pharmaceuticals and cosmetics.

A produção de proteases e biossurfactantes por Bacillus licheniformis UCP-1014 foi investigada neste trabalho. Os experimentos foram realizados em frascos de Erlenmeyer de 125 mL, em triplicata, inóculo 10% v/v, a 150 rpm e 37C. Um planejamento fatorial foi realizado para investigar as concentrações dos componentes do meio de cultivo. Amostras de líquido metabólico foram coletadas, centrifugadas e os sobrenadantes utilizados para determinar pH, atividade proteolítica e tensão superficial. O líquido metabólico foi concentrado por ultrafiltração e a estabilidade da atividade proteolítica no retentado foi determinada quanto ao pH e à temperatura. A estabilidade do retentado foi investigada por planejamento fatorial e a atividade proteolítica determinada com 10, 20 e 30 dias de armazenamento a 28 C. A determinação de proteases foi realizada na presença de azo-caseína. A cultura de B. licheniformis UCP-1014 produziu 112 U/mL de proteases na presença de melaço 1% e uréia 0,5%, a pH 7,5 com 24 h de cultivo. A redução da tensão superficial do líquido metabólico não foi significativa nessas condições de trabalho. O líquido metabólico concentrado reteve cerca de 50% da atividade proteolítica inicial. O concentrado de proteases apresentou a maior atividade enzimática em pH 8 durante 30 min de incubação, retendo 97 % da atividade; a estabilidade térmica máxima foi a 50C durante 30 min, retendo 98 % da atividade enzimática. O retentado do líquido metabólico após formulado manteve 54 % da atividade com 30 dias de armazenamento a 28C. Proteases produzidas por B. licheniformis UCP-1014 na presença de nutrientes de baixo custo podem ser competitivas no mercado
The production of protease and biosurfactant by Bacillus licheniformis UCP-1014 was investigated in this work. The experiments were performed in Erlenmeyer flasks, in triplicate, and inoculum 10% v/v, 150 rpm and 37C. A factorial design was conducted to investigate the concentrations of the medium. Metabolic fluid samples were collected, centrifuged and the supernatant used to determine pH, proteolytic activity and surface tension. The liquid was concentrated by ultrafiltration metabolic the stability and proteolytic activity in the retentate was determined for pH and temperature. In making the retentate was used a factorial design, and protease stability was determined during 10, 20 and 30 days at 28C. The determination of protease was performed in the presence of azo-casein. The culture of B.licheniformis UCP-1014 produced 112 U/mL protease in the presence of 1% molasses and urea 0,5%, pH 7,5 at 24h of culture. The reduction in surface tension was not significant in these metabolic conditions. The concentration of proteases produced by B. licheniformis UCP-1014 had the highest stability of enzyme activity in the absence of substrate at pH 7 during 60 min of incubation and maximum thermal stability between 40 90C for 90 min. The liquid concentrate and formulated metabolic retained about 50% of proteolytic activity whose value decreased during storage at 28C. Proteases produced by B. licheniformis UCP-1014 in the presence of nutrients of low cost can be competitive in the market

碩士
東海大學
化學工程與材料工程學系
101
Antrodia cinnamomea is an endemic medical mushroom in Taiwan. It is well known that the major effective components in this medical fungus are polysaccharides, triterpenoids and steroids. The bioactive and efficacy ofA. cinnamomea are still the main focus in many researches. In this research, the main idea of submerged cultivation of A. cinnamomeais by reusing thin stillage to study their effects on cell growth and the formation of bioactive components. The results showed that when homogenized mycelia were used for inoculation in seed culture, biomass reached high concentration of 2.76 g/L in six days, which was twice more than non-homogenization.Using the same method cultured with time course, non-homogenization could lead to the formation of pellet with big particle size and high crude triterpenoid content. The highest content of totalcrude triterpenoid reached to 19.30 mg/L at 34th days, which was 3.52 times more than non-homogenization. Concerning the effect of inoculum sizes, inoculum 1 % could obtain the biggest particle size and the highest content of total crude triterpenoid of 47.08 mg/L. On the other hand, inoculum 10 % had the smallest particle size and the highest content of total intracellular polysaccharidesof 757.28 mg/L. Compared with the control, polymer addition could lead the rise of the formation of bioactive components. Agar was proved to be more effective than CMC. In the cultures using stirred tank bioreactor, high speed stirring was beneficial to the formation of intracellular polysaccharides.Homogenized seed culture using flask could increase the amount of triterpenoids. Higher inoculum size of 10 % was demonstrated to enhance the formation of triterpenoids and extracellular polysaccharides. In the culture using an air-lift bioreactor with inoculum 10%,mycelium pellets was found and the unit of triterpenoids could increase to 5.95 mg / g. This study also proved that the reuse could reduce the COD values dramatically from 21160 to 2080 ppm.

碩士
靜宜大學
食品營養學系
106
Phellinus linteus is a traditional medicinal mushroom that has been widely used in East Asia. However, the wild resources of P. linteus gradually decreased, and the cultivation of fruiting bodies is labor-intensive and time-consuming. Therefore, mycelium production by submerged cultivation was an alternative source of P. linteus. Many studies have found a variety of compounds from fruiting bodies, fermentation broth and mycelium of P. linteus. Hispidin is one of the attractive metabolites with biologically activities. Many studies focused on the biological activities of hispidin, but studies on hispidin production by submerged cultivation were still rare. The purpose of this study was to optimize medium components and culture conditions for enhancing hispidin production by submerged cultivation of P. linteus. For screening of carbon and nitrogen source of medium, Petri dish and shake flask cultures with different combination of carbon and nitrogen sources were carried out. It was found that the most suitable carbon and nitrogen source for hispidin production was glucose and soybean peptone, respectively. Then central composite design was used to optimize culture time and concentrations of glucose and soybean peptone for hispidin production. The optimal culture time, glucose and soybean peptone concentrations were determined to be 8 days, 36.93 g/L and 5.06 g/L, respectively. Under these optimal conditions, hispidin production in shake flask culture reached 552.09 mg/L, which was about 4 times higher than that of original conditions. Effects of operating conditions in shake flask culture on hispidin production were also studied . In the medium with initial pH 7, hispidin production was higher than that with pH4, 5, and 6. In addition, non-woven cloth, reticulated polyurethane foam, natural loofah were used in shake flask culture for cell immobilization. It was found that P.linteus mycelium could grow and adhere on these porous supports, but hispidin production was lower than that of cell suspension culture. The ratio of gas-liquid interface area (A) to liquid volume (V) of shake flask influenced oxygen transfer rate in shake flask culture. The results showed that an moderate A/V ratio was required, but not too high or too low. Finally, a 5-liter stirred-tank fermentor stirred-tank fermentor was performed for scale-up test. The hispidin production reached 562.78 mg/L after 7 days at an agitation speed of 250 rpm, aeration rate of 0.33 vvm. Hence, hispidin production by submerged cultivation of P. linteus has been scaled up successfully from shake flask to 5-liter stirred-tank fermentor.

碩士
朝陽科技大學
應用化學系生化科技碩博士班
103
Antrodia cinnamomea is an unique wild fungus in Taiwan, which can only grow on the unique Cinnamomum kanehirae basswood. Its fruiting body grows very slowly. In this study, we focus at production of 4-acetylantroquinonol B (4-AAQB) which is a natural compound containing in A. Cinnamomea. 4-AAQB has been reported of anti-proliferative activity on hepatoma cell HepG2. In this study, a submerged cultivation of A. Cinnamomea was performed. Th ethyl acetate (EtOAc) extract of the fermentation broth was analyzed using MALDI-TOF/MS to identify the existence of 4-AAQB in fermentation broth. Further quantification method is now under development. Preliminary results have been consistent with dish culture of basswood color category fruiting bodies, Liquid fermentation 4-AAQB four kinds of conditions, C condition are the best available preferred 4-AAQB content.

博士
國立中興大學
食品暨應用生物科技學系
94
The research reported herein is designed to study the submerged culture of Coprinus comatus and Sparassis crispa to produce high contents dry biomass and polysaccharide of mycelia. Therefore, the evaluation of quality properties, antioxidant properties, and tumor cytotoxicity of mycelia and fermentation filtrate. Furthermore, the evaluation physicochemical properties of hot water and alkali extracts polysaccharides, and tumor cytotoxicity. Coprinus comatus (Muller: Fries) S. F. Gray (Coprinaceae), the shaggy mane or chicken drumstick mushroom and also known as the lawyer’s wig mushroom, is a newly cultivated edible and medicinal mushroom. C. comatus have been reported to lower blood glucose, immunomodulating, antitumor activity, antibacterial activity, antivirus activity, antimutagenic effect. Sparassis crispa (Wulf: Fries), also called cauliflower fungus, is a newly cultivated mushroom and looks like a cauliflower or coral. Recent research indicated β-(1,3)-D-glucan could be extracted from its fruiting bodies, which exhibited the effects of anti-tumor and immunoregulation. Growing mushroom mycelium in liquid culture on a defined nutrient broth has been a simple and fast alternative method to produce fungal biomass. The research reported herein was to study the optimal conditions for submerged culture of C. comatus and S. crispa, to evaluate the dry biomass and polysaccharides of their mycelia and fermentation filtrate. With regard to C. comatus submerged culture, the optimal conditions were pH 5.0, 20°C, 6 days and 100 rpm with carbon and nitrogen sources being 2% fructose and 0.5% yeast extract. Contents of dry biomass and polysaccharides from C. comatus were 7.64 and 0.58 g/L. With regard to S. crispa submerged culture, the optimal conditions were pH 4.0, 20°C, 6 days and 100 rpm with carbon and nitrogen sources being 2% glucose and 0.5% yeast extract. Contents of dry biomass and polysaccharides from S. crispa were 8.73 and 0.45 g/L. Furthermore, the non-volatile components and physiological activity in the two forms of C. comatus and S. crispa mycelia and filtrate were studied. Both mycelia and filtrate of C. comatus and S. crispa were high in contents of carbohydrate. Content of total sugars and polyols were 111.84, 523.61, 61.37 and 556.73 mg/g for C. comatus mycelia, C. comatus filtrate, S. crispa mycelia and S. crispa filtrate, respectively. Glucose contents were the highest in both C. comatus and S. crispa filtrate were 445.44 and 494.75 mg/g, respectively. Contents of total free amino acids were 18.03, 91.13, 24.41 and 81.78 mg/g for C. comatus mycelia, C. comatus filtrate, S. crispa mycelia and S. crispa filtrate, respectively. The contents of monosodium glutamate (MSG)-like components from C. comatus and S. crispa in filtrate (15.81 and 17.73 mg/g) were higher than that in mycelia (3.44 and 3.91 mg/g). The contents of flavor 5''-nucleotides from C. comatus and S. crispa in filtrate (2.71 and 3.20 mg/g) were higher than that in mycelia (1.72 and 2.20 mg/g). EUC values were 52, 415, 70 and 315 g MSG/100 g for C. comatus mycelia, C. comatus filtrate, S. crispa mycelia and S. crispa filtrate, respectively. Overall, filtrate of C. comatus and S. crispa possessed highly intense umami taste. In addition, to investigate the antioxidant properties of ethanolic and hot water extracts of mycelia and filtrate from C. comatus and S. crispa mycelia and filtrate, including antioxidant activity, reducing power, scavenging abilities on radicals and chelating abilities on metal ions. The contents of potential antioxidant components in these extracts were also determined. Hot water extracts were more effective than ethanolic extracts from C. comatus mycelia in antioxidant activity, reducing power, scavenging ability on DPPH and hydroxyl radicals as evidenced by lower EC50 values. Hot water extracts were more effective than ethanolic extracts from C. comatus filtrate in antioxidant activity, reducing power, scavenging ability on hydroxyl radicals and chelating ability on ferrous ions as evidenced by lower EC50 values. Overall, for both extracts, hot water extracts from C. comatus mycelia were more effective among antioxidant properties assayed. Ethanolic extracts were more effective than hot water extracts from S. cirpsa mycelia in antioxidant activity, reducing power, and scavenging ability on DPPH as evidenced by lower EC50 values. Hot water extracts were more effective than ethanolic extracts from S. crispa filtrate in antioxidant activity, scavenging ability on hydroxyl radicals and chelating ability on ferrous ions as evidenced by lower EC50 values. Overall, for both extracts, ethanolic extracts from S. crispa mycelia were more effective among antioxidant properties assayed. With regard to the hot water or alkali extraction from C. comatus and S. crispa fruit body, mycelia and filtrate, the yield was the highest in both mushrooms for mycelia polysaccharides. However, the highest total sugar contents were found in hot water and alkali extracts polysaccharides from filtrate. The microprotein contents of alkali extracts from C. comatus and S. crispa were higher than that of their hot water extracts polysaccharides. In elemental analysis, nitrogen, carbon and hydrogen contents of alkali extracts polysaccharides from C. comatus and S. crispa were higher than that of their hot water extracts polysaccharides. Neutral sugars were mannose and glucose for all polysaccharides isolated. Using gel filtration, the molecular weights of hot water extracts from C. comatus and S. crispa were higher than that of their alkali extracts. Besides, the studies of the effect of ethanolic and hot water extracts of C. comatus and S. crispa fruit body and mycelia and polysaccharide therefrom inhabited cancer cell viability were studied using MTT test. Furthermore, evaluation possibility mechanism of induced cytotoxicity in A549 and SVEC cell line by cell cycle analysis. IC50 values in A549 cell line were 0.69, 1.69, 0.24 and 2.58 mg/mL; in HCT116 cell line were 1.72, 1.53, 1.14 and 1.58 mg/mL; in HL-60 cell line were 2.22, 0.83, not detected and 2.29 mg/mL; in MCF-7 cell line were 0.94, 1.80, 1.11 and 3.22 mg/mL; in SK-Hep-1 cell line were 1.25, 1.98, 2.56 and 8.46 mg/mL; in SVEC cell line were 0.88, 1.95, 3.25 and 5.53 mg/mL for fruit body in ethanolic extracts, mycelia in ethanolic extracts, fruit body in alkali extracts polysaccharide, and mycelia in alkali extracts polysaccharide from C. comatus, respectively. IC50 values in A549 cell line was 1.22 mg/mL; in HCT116 cell line was 3.00 mg/mL; in HL-60 cell line was 1.66 mg/mL; in MCF-7 cell line was 2.14 mg/mL; in SK-Hep-1 cell line was 2.85 mg/mL; in SVEC cell line was 1.70 mg/mL for mycelia in ethanolic extracts from S. crispa, respectively. Cell cycle analysis revealed that ethanolic extracts of mycelia from C. comatus induced apoptosis on A549 via G0/G1 cell cycle arrest. Overall, mycelia and filtrate of C. comatus and S. crispa contained abundant nutritional components and essential amino acids and umami components, and bioactive polysaccharides, possessed antioxidant properties and tumor cytotoxicity. Based on the results, it is a high protein, low fat and no cholesterol health food, and is an alternative to food flavoring.

碩士
國立臺灣大學
食品科技研究所
89
Cordycesps sinensis is a fungus parasitizing on the larva of Coleoptera and Lepidoptera. The fungus contains various proteins, amino acids, fatty acids, nucleotides, and polysaccharides. It also contains some minor components, such as cordycepin, adenosine, ergosteryl-D-glucopyranoside, and 2,2-dihydroergosteryl-D-glucopyranoside. In Chinese, the Cordycesps sinensis is considered as a super-effective nutritious food supplement and medicine. Recent researches have also shown that the Cordyceps species can enhance immune system, exhibit anti-tumor effect, and improve cardiovascular diseases. In order to produce this fungus in a large scale, this research project attempted to use fermentation technology to culture the mycelium of Cordyceps species, and conferred the conditions of fermentation. Moreover, in order to understand this fungus further, and to increase the variety of this product, various extraction and separation methods were used to yield various fractions out of the fermentation broth, and the antioxidative activity of each fraction was investigated. 　　During submerged cultivation of Cordyceps sinensis and Cordyceps militaris in a 5L fermentation tank, pH of the broth didn’t change significantly throughout 10 days of cultivation. The yields of mycelium of both species reached maximum at 5 days. The maximum amount of adenosine was produced during the early days (about 2-4 days) of cultivation, and the cordycepin were in the later period of cultivation (after 6 days). In general, one could get better yields of adenosine and cordycepin in the W formula medium, and C. militaris produced more adenosine and cordycepin than that of C. sinensis. 　　The fermentation broth of Cordyceps militaris contained 4.07% crude fats, 5.73% crude proteins, and 1.82% ash. The total amino acid contents in the mycelium and medium (mycelium-free) were 21.25% and 1.39% respectively, including all of the eight essential amino acids, and histidine that infant requires. 　　The mycelium-free medium of C. militaris were separated based on molecular weight differences using membrane technology, and the obtained fractions were tested for antioxidative activities using the methods of reducing power, scavenging ability to superoxide anion, scavenging effects on DPPH, and chelating ability to copper and ferrous ion. It was found that potent antioxidative activities did existed in the fraction with molecular weight less than 3 kD and the fraction between 3 kD and 10 kD. However, no antioxidative activity was detected in the fractions extracted from the mycelium with various solvents.

Aquaculture is expected to play a key role in the increase in fishery production in the future. Because of environmental and coastal utilization issues, however, it is necessary for us to operate cultivation in exposed area for more fishery production, where the fish cage and its mooring system face severe environmental condition such as typhoon. In that area the fish cage should be submerged to protect it from high waves since the influence of waves is reduced in deeper water. Nevertheless, the technologies for open ocean aquaculture are still in the phase of being developed. For example, in general operating systems, a fish cage is floated or submerged on the basis of the framework which is fixed by buoys and anchors. In the exposed area, however, buoys are broken by waves, resulting in the high maintenance cost. Our research aims at creating the new mooring system of a fish cage. The fish cage is moored without the framework in various depths. Buoys can be submerged so that the maintenance cost of the mooring system must be reduced. The feasibility of the mooring system is examined by smaller-scale water tank testing and simple numerical analysis. First the behavior of the mooring system in still water was examined by both numerical analysis and tank model testing. As a result, the fish cage can be submerged at an arbitrary depth only by changing the ratio of the buoyancy to the weight of the fish cage. Then, using a new model of a fish cage, towing test was carried out to estimate the drag force. The drag force is proportional to the 1.5 powered velocity of water current, which is different from the second powered velocity usually used in numerical analysis. This discrepancy may be attributed to the lack of measured data. It should be also noted change in the attacking angle of water current can influence the drag force. Finally, towing test of the mooring system was carried out at the various towing speeds to examine the change in the geometrical formation of the system. At the higher towing speed fish cage is more submerged due to the increase of drag force. In addition, the fish cage begins inclining just after towing. The drag force must vary since the projected area changes due to the inclination of the fish cage and to the deformation of the net. However, the forces on the fish cage are balanced again so that the geometrical formation becomes stable. As future studies, the experimental result will be compared with the analytical one, in which the effect of drag force will be added to the balance of forces on the fish cage. The water tank has a wave maker, so waves will be added to the water current since wave-induced forces work on the fish cage submerged near water surface.