Dissertations / Theses on the topic 'Submerged cultivation'

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.

碩士
朝陽科技大學
生物技術研究所
94
Antrodia camphorata is a peculiar and extremely expensive medical fungi in Taiwan. Recent studies on these fungi shown to have various biological activities including modulation of immune response and inhibition of tumor growth. Now submerged cultures can produce a large number of mycelium under short time and limited space, so it is still the major mode to culture and have commercial economic development ability very much. In this study, we used submerged cultivation to produce the mycelium, and its extraction and chemical purification from the different strains i.e. BCRC 35396, BCRC 35398 and BCRC 35716 of A. camphorata. The results indicated that the strain BCRC 35398 is better than the others where the extracellular polysaccharides concentration is 1.01 mg/mL, mycelium dry weight is 1.95 mg/mL, cell number is 6.2×105 and the (1→3)-β-D-glucan is 0.29 mg/mL, respectively. Although, the BCRC 35398 strain is better than the others, however, BCRC 35398’s (1→3)-β-D-glucan transform rate is better. We found that the BCRC 35398 produced extracellular polysaccharide has molecular weight is 1,425,633 and the percentage is 3.1 % in total carbohydrate by using gel filtration chromatography and dyeing method. Through the chemical extraction method, we found that (1→3)-β-D-glucan is the curdlan. In the 13C-NMR spectra, the chemical shift values of (1→3)-β-D-glucan were as follows: C-1 at 103.140 ppm, C-2 at 76.415 ppm, C-3 at 86.250 ppm, C-4 at 68.490 ppm, C-5 at 76.415 ppm and C-6 at 60.958 ppm, respectively.

碩士
國立屏東科技大學
熱帶農業暨國際合作系所
98
The edible mushroom Grifola frondosa is a Basidiomycete fungus belonging to the order Aphyllopherales, and family Polyporaceaehas. Polysaccharides extracted from the mushroom have been reported to have antitumor and immunomodulating activities. Other medical uses of the mushroom include antioxidant, antidiabetic, and cardiovascular activities. In the present research, G. frondosa was cultivated in shaking flasks and investigated the effects of agitation speeds, inoculum ratios, and initial pH on the pellet morphology, biomass, and extracellular polysaccharide production. Furthermore, mathematical models for optimization of the three factors for biomass and extracellular polysaccharide production were developed. In addition, immunomodulating activities of extracellular polysaccharides produced by submerged cultivation under optimized conditions was also evaluated. The results showed that the culture parameters including agitation speed, inoculum ratio, and initial pH influenced pellet morphology, biomass, and extracellular polysaccharide production. Low agitation speed at 100 rpm resulted in small and hairy pellets but greater amount of biomass and extracellular polysaccharides which were 5.30 mg/ml and 2.14 mg/ml, respectively, while higher agitation speed at 150 rpm led to larger pellet size but less biomass and extracellular polysaccharides which were 5.03 mg/ml and 1.62 mg/ml, respectively. On the other hand, less inoculum ratio 2% led to smaller pellet size and less amount of biomass 4.51 mg/ml while maximum extracellular polysaccharide production 2.46 mg/ml, whereas 4% of inoculum resulted in larger pellet size and greater amount of biomass production 5.21 mg/ml but less amount of extracellular polysaccharides 1.68 mg/ml. In addition, initial pH 5.5 and 6.0 were favorable for both biomass and extracellular polysaccharide production. The optimal conditions for biomass production were agitation speed at 105 rpm, inoculum ratio 3.1%, and initial pH 5.5. Under this condition, the predicted maximum biomass was 5.28 mg/ml. Regarding to optimization of extracellular polysaccharide production, the optimal conditions were agitation speed at 111 rpm, inoculum ratio 3.3%, and initial pH 5.6. Under this situation, the predicted maximum extracellular polysaccharides was 2.35 mg/ml. HPLC-RI results indicated that, two extracellular polysaccharides with molecular weight of 21.16 and 1.69 kDa were identified. The polysaccharides showed immunomodulating activity by increasing the proliferation of peripheral blood mononuclear cells. Optical density at 450nm of the treatment with polysaccharide from G. frondosa was 1.12 higher than that of control and LPS treatments which were 0.74 and 0.79, respectively. The findings may be useful in future for development of proper fermentation techniques for production of G. frondosa products.

碩士
亞洲大學
生物科技學系碩士班
99
Grifola frondosa is one kind of medical and edible fungus, and it has been found lots of functions such as: anti-tumor, strengthen the immune system, lowering blood glucose, blood pressure and cholesterol, and weight-loss and other anti-diabetic effects. The international biological and medical community has been highly paid attention to G. frondosa recently. Due to the traditional cultivation of G. frondosa from the inoculation to produce fruiting bodies which takes a long time, the fermentation technology to produce mycelium and polysaccharides can not only shorten the training time, but also increase the quality and the yield stably. For the above reasons, the three purposes of this study are as follows. (1) To investigate the best carbon and nitrogen source of the five strains of G. frondosa by solid-state culture. (2) To study the production effects of G. frondosa mycelium and polysaccharide by different rpm and different initial pH value of submerged culture. (3) To analyze total polyphenol and flavonoid content and fermentation DPPH radical scavenging ability of the fermentation broth of G. frondosa. The first part of experimental results showed that best carbon source and the nitrogen source for the mycelium growth of five strains G. frondosa after 20 days cultivation were as follows: the 4% fructose as the carbon source and the 2% corn steep powder as the nitrogen source caused the mycelium growth of T1 strain reached 40.17 mm; the 3% glucose and the 1.5% corn steep powder let the mycelium growth of T2 strain reached 42 mm; the 4% fructose and the 1.5% yeast extract urged the mycelium growth of BT strain reached 42.83 mm; the 4% glucose and the 1.5% corn steep powder caused the mycelium growth of D-4 strain reached 41 mm; the 4% glucose and the 1.5% corn steep powder caused the mycelium growth of 5-T strain reached 42 mm. The second part of the study was designed to investigate the different rpm and initial pH value on the impact of submerged culture of G. frondosa. According to the experimental results, All strains (T1, T2, BT, D-4 ,5-T) in rpm 150 under cultivation, the highest yield of polysaccharide were 2.82±0.56, 2.11±0.13, 3.24± 0.71, 2.85±0.39, 2.06±1.23 g/L, but the mycelium and the final pH of no significant impact of the change. Part of the initial pH value, T1, T2 and BT strains of G. frondosa, produced respectively the highest mycelial yields as 1.83 g/L, 4.18 g/L and 3.16 g/L when the initial pH value was 5.5. In addition, D- 4 and 5-T strains, produced the highest mycelial yields when the initial pH values were 6.5 (3.83 g/L) and 4.5 (3.14 g/L). Furthermore, the maximum polysaccharide production of G. frondosa T1, T2, BT and 5-T strains were 8.39, 7.57, 5.56 and 5.07 g/L when the initial pH was 4.5. The D-4 strain presented the highest yield of polysaccharides (7.07 g/L) when the initial pH was 6.5. The third part of the antioxidant ability experiments indicated that the fermentation broth of T2 strain contained the highest total polyphenols (29.75 mg/g) and flavonoids (1.20 mg/g) above other strains under the cultivation conditions of 3% fructose and 1.5% corn steep powder at initial pH of 6.5. Moreover, T1, T2, BT, D-4, and 5-T strains presented the highest ability of scavenging DPPH radicals were 53.70%, 46.88%, 57.81%, 48.68% and 55.54%. Especially, G. frondosa 5-T strain presented the best results of EC50 within five strains was 23.67 mg/mL under the fermentation conditions of 4% glucose, 1.5% corn steep powder and initial pH of 4.5. Summarize the above results, higher concentrations (4~3%) of glucose and fructose as the carbon resources were suitable for the mycelia growth of all five strains of G. frondosa. Lower concentrations of corn steep powder (2~1.5%) as nitrogen resources were suitable for all strains of G. frondosa. The initial pH values (the case of weak acid) of G. frondosa fermentation were not significantly affected the mycelium and polysaccharide productions, but they had a little impact on the antioxidant capacity of G. frondosa. The relationship between different carbon-nitrogen resources and antioxidant abilities; or between different surfactants such as vegetable oil and polysaccharide production of G. frondosa can be further investigated. Based on the achievement of this study, the related information can be provided to the farmers for the solid-state of cultivation and the submerged culture in five different strains of G. frondosa. In the future, the fermentation fluid of G. frondosa can be applied for the development of functional health products to increases the industrial utilities and to promote the additional values of G. frondosa.

碩士
大同大學
化學工程學系(所)
92
The experiments of submerged cultivation of Monascus anka were carried out according to the SPUD design. The control factors were the cultivation temperature and the dissolved oxygen. The measurements of glucoamylase activity and concentration of red pigment from the experiments were used to identify a second-order response model. Eleven experiments located by the initial UD method followed by the SPUD method were used to build the second-order response model with 2.71% average modeling error for the glucoamylase activity but 12.44% average modeling error for the concentration of red pigment. Two additional experiments could not improve the quality of the second-order response model describing the concentration of red pigment. Considering the time resource for carrying out the experiments, the sub-optimal operating condition was determined based on available experimental data to achieve the maximal overall desirability objective function of glucoamylase activity and concentration of red pigment of the end product. The experimental results shown in this work demonstrated the applicability of the SPUD method to locate limited experiments for guiding the experimenter to reach a sub-optimal or optimal operating condition of an unknown process.

碩士
國立中興大學
食品暨應用生物科技學系所
98
Ergothioneine (EGT) is a kind of precious amino acid, it can prevent the oxidative damage and anti-inflammation in organism. EGT exist in plants and animal tissues, it cannot be biosynthesized in human, only can be absorbed from the diet. In recent the research discovered the rich content of EGT in the mushroom, especially the Pleurotus mushroom content higher EGT. Content of EGT in Pleurotus eryngii (1521.6 mg/kg dw) is the highest from the 28 kinds of common mushrooms. Therefore, this research used the submerged fermentation to cultivate the P. eryngii to produce the high EGT content P. eryngii mycelia. In addition, the research also analyzed the proximate components, nutritional components, taste and physiologically active components, antioxidant properties and evaluation of anti-inflammation effect on the fruiting bodies (FB), base of fruiting bodies (BFB) and high EGT content mycelia (HEM) of P. eryngii. Cultivated mushroom mycelium with the liquid culture has lots of benefits content simple, fast alternative, stabled quality and easy to scale up. The research reported to study the optimal conditions for submerged culture of P. eryngii, to increase the EGT content in the P. eryngii mycelia. The result showed that optimal conditions for high content of EGT were inoculated with 10 % (v/v), 2 % glucose as the carbon source, 0.5 % yeast extract as nitrogen source and the cultivated at 25℃ on a shaking incubator at 125 rpm, then added 4 mM Histidine after cultivated 7 days, after cultivated 18 days the EGT content were 48.09 mg/L. Used the same optimal culture to cultivated the P. eryngii mycelia at 10 L fermenter with the following conditions: temperature, 25℃; aeration rate, 1 vvm; agitation speed, 150 rpm, after cultivated 18 days the EGT content of mycelia was 62.20 mg/L. With regard to proximate composition carbohydrate (57.42 %, 59.88 % and 27.81 %) were the major components found in FB, BFB and HEM. With regard of physiologically active components, FB content highest ergosterol, 6.17 mg/g, otherwise the contents of polysaccharide and ergothioneine of HEM were 155.13 mg/g and 3.93 mg/g, higher than FB and BFB. With regard to taste characteristics, the contents of 5’-nucleotide and free amino acid of HEM (20.33 mg/g and 10.56 mg/g) were higher than FB and BFB, however, the content of soluble sugar and the taste characteristics of FB were better than BFB and HEM. On the antioxidant activity, Trolox equivalent antioxidant capacity (TEAC) of HEM (20.59 μmole Trolox/g) was significant higher than FB (13.42 μmole Trolox/g ) and BFB (0.11 μmole Trolox/g). The ethanolic extract from HEM at the concentration of 20 mg/mL, the antioxidant activity (78.79 %), the reducing power (1.92) and the scavenging ability on 1,1-diphenyl-2-picrylhydrazyl radicals (95.02 %) were higher than BFB, furthermore, the total phenol and flavonoid were also rich in HEM than in BFB and FB. In the anti-inflammation effect test, we investigated the effect of hot water and ethanolic extract from FB, BFB and HEM of P. eryngii on LPS-induced NO and TNF-α production in RAW 264.7 cell. The results showed that the ability of inhibition NO and TNF-α production in LPS-induced RAW 264.7 cell the ethanolic extracts were effective than the hot water extracts. The NO content of FB, BFB and HEM were 10.80 μM, 10.99 μM and 10.90 μM, all of that were significant lower than control (15 μM), when the ethanolic extracts concentration was 1000 μg/mL, and when the concentration were 500 μg/mL, the TNF-α content of FB, BFB and HEM were 3.05 ng/mL, 3.59 ng/mL and 2.78 ng/mL, that were also significant lower than control. The results suggest the ethanolic extracts from FB, BFB adn HEM from P. eryngii have an anti-inflammatory activity. Overall, added amino acid (histidine, cysteine and methionine) could increase the EGT content of P. eryngii mycelia, and the HEM was rich in nutritional component, essential amino acids, umami components, physiologically active components, antioxidant activity and anti-inflammation activity.

博士
國立臺灣大學
食品科技研究所
96
The objective of this research was to establish proper fermentation conditions for submerged cultivation of Antrodia cinnamomea (AC) to produce a fermentation product with antihepatoma activity. AC was fermented for 1, 2, 4, 6 and 8 weeks with galactose, lactose and glucose as carbon sources of the submerged cultivation medium in 250 ml flask. The cell viability of Hep3B was more inhibited with the treatment of ethanolic extract of AC mycelia fermented for 8 weeks than those fermented for 1, 2, 4 and 6 weeks. The IC50 of ethanolic extract of AC mycelia fermented for 8 weeks with glucose as a carbon source (AC8-Em-glu) was 76.2

碩士
大葉大學
生物產業科技學系
94
Bacillus subtilis was used as a biocontrol agent to suppress plant fungal pathogens. The secondary metabiolite, iturin A, which produced by B. subtilis consists of a peptide ring of seven amino acids. The iturin A exhibits a strong antifungal, and antibacterial activity as well as biosurfactant property. The applications of this compound have been limited due to its poor production yield.　The purpose of this research was to study the optima production of iturin A of B. subtilis by using the response surface method (RSM) in submerged cultural fermentation. In the shake flask study, the glucose was found to be the best carbon source for the concentration of iturin A among several carbon sources including glucose, fructose, sucrose and maltose. On the other hand, the corn steep powder was the better nitrogen source in the selected testing nitrogen source such as soybean albumen, yeast powder, peptone special and corn steep powder. In the mutual experiment of potassium phosphate and magnesium sulfate, we found that magnesium sulfate has the apparent effect to improve the production of iturin A. the optima compositions of iturin A from the RSM was pH 4.5, 0.93% dextri-maltose, 1.11% glucose, 0.72% C.S.P, 1.5 mM MgSO4, 0.75 mM KH2PO4, rotation speed 181 rpm and area of aeration 4.35 cm2.　By using the 5-L fermentor, it is help of the growth of B. subtilis and production of iturin A that improve rotational speed. In the experiment with different air flow rate, there is better production of iturin A in high aeration of 2 vvm. In the experiment with different baffles, there have the batter KLa and the best production of iturin A (about 112.12 mg/L) when adding baffle 1 in the fermentor.

碩士
國立中興大學
化學工程學系所
103
Antrodia cinnamomea is an endemic species of fungi in Taiwan. Previous reports indicated that fruiting body of A. cinnamomea is characterized by varied physiological active compounds such as polysaccharrides, triterpenes, sesquiterpenes, and steroids et al, which proved to be effective for anti-oxidation, anti-tumor,anti-inflammatory, hepatoprotective and antihypertension treatments. Traditional Chinese herbs, such as Gastrodia elata, Mesona chinensis and Scutellaria baicalensis have been reported having the function of reducing blood pressure. In this project, the submerged culture of A.cinnamomea was carried out associated with the addition of these three Chinese herbal extracts, in order to elevate the ACEI activity of A.cinnamomea. In carbon source test, glucose gave had the highest biomass and ACE inhibition. In the nitrogen source test, yeast extract yeilded the highest biomass at eighth-day and peptone as nitrogen source produced the highest ACE inhibition, which was equal to 4929.5 ng captopril/L. The addition of Chinese herbal extracts to A.cinnamomea cultivation was conducted. The highest biomass had 13.59 gDW/L by adding 10g/L of Gastrodia elata ectracts. The highest ACE inhibition was equal to 8202.1 ng captopril/L by adding 10g/L of Mesona chinensis extracts, which was 3.2 times higher than the control test. However, the biomass and ACE inhibition were both decreased by adding different concentration of Scutellaria baicalensis extracts.

碩士
國立宜蘭大學
食品科學系碩士班
101
Sorghum distillery residues (SDR) is large and cheap by-product in brewery, but it still contains nutrients and bioactive components. It was used in livestock or poultry fodder. Coriolus versicolor (syn. Trametes versicolor, Yunzhi), a white rot fungus found worldwide, is a medicinal mushroom with a wide applications and the fungus can be grown in submerged fermentation as mycelial biomass. The purpose of this study was to investigate the characteristics of intracellular and extracellular polysaccharides, and antioxidant capacity, physiologically active components of mycelial of C. versicolor cultured in sorghum distillery residues liquid medium at 25 °C, 100 rpm in shake flask in different culture days. Submerged culture C. versicolor with sorghum distillery residue liquid culture medium, we obtained 5.69 g/L extracellular polysaccharides after 7 days and 0.42% (1-3)-glucan in extracellular polysaccharides after 11 days. In addition, we obtained 3.51 g/L intracellular polysaccharides and 25.35 g/L biomass after 11 days. The content of (1-3)-glucan had no significantly different during 5-13 days, that were 0.31%-0.35%. Observed the molecular weight distribution of intracellular and extracellular polysaccharide in C. versicolor during cultured, the percentage of the large molecular weight area (I, Mw>110 KDa) of extracellular polysaccharide in C. versicolor were increased dependent on cultured days. The extracellular polysaccharide have the highest molecular weight after cultured 7 days (Mw>670 KDa, retention time were 5.03 min). However, in the late stage of culture, the molecular weight of large molecular weight area of extracellular polysaccharides in C. versicolor will slightly decline. The percentage of the large molecular weight area (I, Mw>110 KDa) of C. versicolor intracellular polysaccharides were also increase dependent on cultured days. The intracellular polysaccharide have the highest molecular weight after cultured 7days (Mw>670 KDa, retention time were 4.45 mins). Moreover, the extracellular polysaccharides after 5 and 13 cultured days and the intracellular polysaccharides after 5, 7 and 9 cultured days were polysaccharide peptide or polysaccharide-protein complexes. About of antioxidant capacity of intracellular and extracellular polysaccharides in C. versicolor after cultivation were appeared antioxidant capacity. Between the antioxidant capacity of polysaccharide in C. versicolor for different culture days, just extracellular polysaccharide cultured to 11 and 13 days possessed significantly higher in DPPH radical scavenging ability, but the capacity was less than trolox. Besides, between the DPPH radical scavenging ability of methanol extracts, from those of C. versicolor of mycelium, fermentation fluid and sorghum distillery residue fermented, the best ability was that of sorghum distillery residue fermented after cultured 5 days. The reducing power of methanol extract of the fermentation fluid was strongest. The total phenolic content, total flavonoid content and the triterpenoids substances content of sorghum distillery residue fermented after cultured 5 to 11 days were higher than those in mycelium and fermentation fluid, and significantly higher than sorghum distillery residue after cultured 13 days.

碩士
南台科技大學
生物科技系
97
This study is concerned with optimization of submerged culture conditions for exo-biopolymer production by one-factor-at-a-time and Taguchi methods and rotating simplex method. The one-factor-at-a-time method was adopted to investigate the effects of medium components (i.e. carbon, nitrogen, and mineral sources) and environmental factors (i.e.initial pH) on mycelial growth and exo-biopolymer production. Among these variables, glucose, yeast extract were identified to be the most suitable carbon, and nitrogen, respectively. The optimal initial pH for mycelial growth and exo-biopolymer production was 5.0, respectively. Subsequently, the concentration of glucose, yeast extract, KH2PO4 and MgSO4．7H2O were optimized using the Taguchi method. The effects of media composition on the mycelial growth of Paecilomyces farinosus were in the order of glucose﹥yeast extract ﹥KH2PO4 ﹥MgSO4．7H2O, and those on exo-biopolymer production were in the order of glucose﹥KH2PO4 ﹥MgSO4．7H2O ﹥yeast extract. The optimal concentration of media components were determined as glucose 3.0 %, yeast extract 0.5 %, KH2PO4 0.05 % and MgSO4．7H2O 0.15 % for the production of mycelial yield and glucose 3.0 % 、yeast extract 0.4 %、KH2PO4 0.1 % and MgSO4．7H2O 0.15 % for the production of exo-polysaccharide, respectively. The subsequent verification experiments confirmed the validity of the models. According to the optimal exopolysaccharides concentration of the Taguchi method, the maximum exopolysaccharides production reached 0.429 ± 0.018 g/L. Finally, rotating simplex method was employed to approach the best combination of physical parameters (viz. pH, agitation rate, aeration rate, temperature) for maximun exopolysaccharides production by P. farinosus in 5L fermentor. The optimal combination was determined to be a pH of 3.33, an aeration of 1.22 vvm, an agitation of 93 rpm, a temperature of 27.67 ℃, producing a 3.5-time increase in exopolysaccharides production (1.454 ± 0.004 g/L) when compared with that achieved in Taguchi Orthogonal arrays.

碩士
南台科技大學
生物科技系
95
Beauveria amorpha is an entomogenous fungus noteworthy for its various bioactivities. The influence of medium and cultural conditions on polysaccharides production was investigated in shake flask culture. The optimal temperatures for both mycelia biomass and EPS production was 26℃, and corresponding optimal initial pHs were found to be 8 and 5, respectively. The suitable carbon and nitrogen compositions of culture medium for the EPS production were sucrose and yeast extract powder, respectively. The highest EPS production (0.477 ± 0.047 mg/ mL) was achieved in a medium of 75 g/L sucrose, 5 g/L yeast extract powder at 26℃, and an initial pH 5.0 in shake flask culture. Under the suggested culture conditions, the maximum concentration of EPS was 0.746 ± 0.038 mg/ mL in a 5-L stirred-tank fermenter. The macrophage cells (Raw264.7) can be stimulated by exo-polysaccharide isolated from the fermentation broth to secrete cytokines such as TNF-α, IL-1β, and IL-6. The results also indicated that the various degree of responsible strength from the macrophage was stimulated by the exo-polysaccharide form the different fermented medium.

碩士
東海大學
化學工程與材料工程學系
105
Cordyceps militaris has been used as a substitute of Cordyceps sinensis. Its demand is quickly rising in the whole world. The biological activities, like anti-inflammatory, antioxidant, anti-aging, antitumor and immunoregulation have been well known. Two of main active ingredients are primary metabolite (adenosine) and secondary metabolites (cordycepin). In this study, different culture strategies and additive were tested to investigate their influence on the formation of bioactive components in submerged cultures of Cordyceps militaris. The main experiment items included static culture with fed-batch, two-stage culture with fed-batch operation, and repeated batch culture. 　　In the tests of static cultures or two-stage cultures with fed-batch operation the addition of glucose and yeast extract achieved higher biomass and primary metabolite (adenosine). The levels of biomass concentration reached 30.72 (g/L) and 26.29 (g/L) on day 31, respectively and the corresponding adenosine concentration were 36.91 (mg/L) and 64.89 (mg/L) on day 17. However, adding soy peptide could obtain higher cordycepin concentration, which were 580.46 (mg/L) and 706.85 (mg/L) on day 31, respectively. Among various culture strategies, contents of adenosine and cordycepin in two-stage culture with fed-batch operation were higher than the static culture with fed-batch operation. So the strategy of two-stage culture is superior to that of static culture for the production of the active ingredients of Cordyceps militaris. 　　The tests of repeated batch culture were performed in Erlenmeyer flask in either shaking or static ways. The static repeated batch culture was demonstrated to be better, in which the concentration of cordycepin reached to 895.25 (μg/ml) in second cycle, and was 1.55 times more than the first cycle. The strategy of repeated batch culture was proved to be effective for enhancing the active ingredients cordycepin of Cordyceps militaris. The results of this study provide the practicable strategies and additives for improving the active ingredients of Cordyceps militaris. Keywords: Cordyceps militaris, static culture, two stage culture, repeated batch culture, cordycepin.

碩士
國立中興大學
食品暨應用生物科技學系所
101
A hemicellulase-producing fungal isolate, Aureobasidium pullulans NCH-218, isolated from our lab（Huang, 2012） was used in the study. The purpose of the study was to produce, purify and characterize the mannanase from A. pullulans NCH-218. The optimal medium and cultivation conditions for A. pullulans NCH-218 was determined in shaking flask（capacity 50mL/250 mL）. The results showed that 3% soybean okara was used as basal medium with 1% locust bean gum as additional carbon source, 0.4% (NH4)2SO4 as additional nitrogen source. Incubation temperature at 30℃, initial pH at 5.4-5.5, inoculum size 1%（v/v）（1.0 × 105 cell/mL）, and shaking rate at 130 rpm gave the best result for the enzyme production. The maximum activity approximately 3.55 U/mL in the culture broth was obtained after 3 days under the above conditions. The broth filtrate from A. pullulans NCH-218 was subsequently purified by ultrafiltration, ammonium sulfate precipitation（60-80%） and DEAE-Sepharose Fast Flow ion exchange chromatography. The nearly purified mannanase had molecular weight around 34 kDa. The optimum pH was pH 3.0, while the enzyme remained quite stable in pH ranging pH from 3.0 to 6.0. The optimum temperature was 60℃, and the enzyme was stable at temperature under 40℃. The purified mannanase demonstrate a specificity towards mannans-based substrates. The hydrolysis products from locust bean gum was manno-oligosaccharides, mainly mannobiose and mannotriose.

碩士
國立臺灣大學
食品科技研究所
92
The objective of this research was to study the fermentation conditions for submerged cultivation of G. lucidum in a 5L fermenter using the medium containing leguminous plants. After the fermentation conditions were established the anti-allergic effects of the fermented products were investigated using cell culture systems. It was found that cultivation of G. lucidum in the medium consisting of 5% black soybean, 2% Astragalus membranaceus (Huangqi), and 2% glucose at 24℃, agitation speed 50 rpm, and aeration rate 0.5 vvm, yielded the highest crude triterpenes (31.82 mg/g D.W.). However, the fermentation conducted at 0.75 vvm and 18℃ yielded the highest amount of (1→3)-β-D-glucan. The maximum amount of aglycone was obtained on Day 5 during fermentation at an aeration rate 0.25 vvm and 0.75 vvm. The peptides contents increased during fermentation with time, and the content of total polyphenols reached a maximum value on Day 5, and decreased thereafter. For the anti-allergic effect, the mononuclear cells (MNCs) from human and EL4 cell from mouse were used. Results indicated that the fermented products could reduce the secretion of TH1/TH2-type cytokine (IFN-γ、IL-2、IL-4) by the mitogen-activated MNCs and EL4 cells. And the fermented products obtained at 24℃ and aeration rates of 0.5 vvm and 0.75 vvm had the potential to become a health-promoting food ingredients with anti-allergic function.

碩士
國立嘉義大學
生物科技研究所
93
Two strains of Escherichia coli (BCRC 10675) and E. coli O157:H7 (BCRC 15374) were submerged cultivated with Tryptic soy broth (TSB) and TSB supplemented with 5% ethanol, respectively. During cultivation, broth turbidities and pH values were determined periodically. The growth rate shown by increase of turbidity for both cultures was retarded significantly by ethanol supplementation. The times transited from log phase to stationary phase were delayed 7 and 14 h for both cultures as affected by ethanol supplementation. When cultivated in TSB, broth pH values decreased from 7.18 to 5.96 in 5 and 6 h and increased to 6.02 in 9 h of incubation for E. coli and E. coli O157:H7, respectively. When cultivated with ethanol, broth pH values decreased from 7.19 to 5.54 and 5.63 in 12 and 20 h of incubation and increased to 5.62 and 5.76 in 17 and 26 h of incubation for the cultures correspondingly. In the log phase, decreases of broth pH value were closely related to increases of turbidity (growth). When the bacterial cells were respectively harvested from log and stationary phases and subjected to fatty acid extraction, methylation and gas chromatography equipped with HP-1 and Rtx-2330 columns for analysis and GC-MS equipped with HP-5 and Rtx-2330 column for identification, trans-9-octadecenoate (18:1, trans-9), five saturated fatty acids (SFAs), two cyclopropane fatty acids (CFAs) and four cis-unsaturated fatty acids (cUFAs) were detected. Under the conducted condition by using Rtx-2330 column, trans-9-octadecenoate (18:1, trans-9), cis-9-octadecenoate (18:1, ciss-9) and cis-11-octadecenoate (18:1, cis-11) could be resolved for quantitative quantification. As affected by ethanol supplementation for both cultures, in stationary phase, the higher trans-9-octadecenoate contents were observed in the cells harvested from TSB supplemented with 5% ethanol than the counterpart cells harvested from TSB. UFAs/SFAs ratios for the log-phase cells of E. coli and E. coli O157:H7 harvested from TSB and TSB supplemented with ethanol changed from 0.91 to 0.54 and from 0.87 to 0.59, respectively. CFAs contents were also higher in the cells harvested from TSB supplemented with ethanol. Those fatty acid variations due to supplementation of ethanol in the growing broth might result in necessary adaptation of membrane fluidity, permeability and mass transferring efficiency to achieve successful surviving growth under stress of ethanol.

碩士
國立臺灣大學
食品科技研究所
95
Antrodia cinnamomea was a popular folk medicine that has attracted great attention due to its antitumor activity. In the last decade, the triterpenoids were isolated from A. cinnamomea and their biological activities such as anticancer and hepatoprotection have been reported. Elicitors have been effective in inducing the synthesis of secondary metabolites (e.g., triterpenoids) in submerged cultivation of A. cinnamomea. A defense response was induced in the culture when elicitors were added. Elicitors can enhance the secondary metabolites in fungal cells. The objective of this study was to investigate the effects of different elicitors (chitosan, CaCl2 and combination (chitosan+CaCl2)) on the triterpenoids production in submerged cultivation system of A. cinnamomea. It was found that the maximum biomass reached 15.8 g/L and 12.9 g/L in airlift bioreactor with dual-net draft tube and fed-batch (day 8) cultures, respectively. The elicitors study showed that treatment of chitosan (100 mg/L) not only obtained more biomass than combination treatment, but also achieved the highest total triterpenoids production. However, combination seriously affected the permeability of cell membrane and resulted in the loss of cell viability. Although the use of combination was not appropriate for long-term cultivation, it did enhance the fungus to accumulate the intracellular triterpenoids in a short-term cultivation. In addition, feeding CaCl2 had little effect on accumulation of triterpenoids for short time cultivation, so it was suitable for long-term fermentation. For two-stage culture strategies, the results proved that triterpenoids production could be highly enhanced by means of the control of oxygen limitation and temperature-shift strategy. For instance, the maximum triterpenoids production (1615 mg/L) was observed at static culture with temperature fluctuation (TF-S group), which was significantly higher than the control. In vitro anticancer test, the results indicated that the crude triterpenoids from TF-S group at concentration of 28 μg/ml inhibited 50% cell viability of HeLa cells, while exhibiting under 100 μg/ml no significant cytotoxicity to WS1 (normal cells).

This graduation theses was intent on study influence conditions submerged cultivation select types enthomopatogenic fungi in liquid nutritive medium with emphasis on optimalization key elements of the process that manner performance uniform biomass mythosporotic fungi {--} blastospores. Experimental part of work was conceived with regard on next sphere problems: 1.Nutritive soil compositionon effect on production blastospores 2.Comparing possibility different kinds and strains enthomopathogenic fungi produce blastospores in submerged cultivation. 3.Conditions submerged cultivation effect on production and yield blastospores 4.Verify possibility production of uniform biomass blastospores in range usable for large-screen application

碩士
國立交通大學
生物科技研究所
89
Ganoderma lucidum and related species are fungi used in traditional Chinese medicine. Recent studies on this fungus have demonstrated many interesting biological activities, including increasing non-specific immunoactivity. Therefore, this study is carried out to determine the effect of cultivating conditions on the mycelial growth and polysaccharide formation of Ganoderma lucidum in submerged cultures. This research is carried out by “ One factor at a time technique ” and “ Response surface methodology, RSM “. The results of “ One factor at a time technique “ show that olive oil added, medium C/N ratio and medium initial pH have significant effect on Ganoderma lucidum submerged cultures. We study on olive oil added, medium C/N ratio and medium initial pH for optimization of polysaccharide formation of Ganoderma lucidum by RSM technique. The optimal olive oil added, medium C/N ratio and medium initial pH are 0.35~0.55mL, 45~55 and 4~5.

碩士
實踐大學
食品營養與保健生技學系碩士班
97
Antrodia cinnamomea is a unique medicinal fungus that has been used as a functional food in Taiwan. The concepts of this study are investigating the physiological and metabolic changes through observing the proteome of A. cinnamomea BCRC 35396 while it is fermenting. In order to compare the expression of proteins among each growth phase, we constructed 2-DE patterns of intercellular proteins from each growth phase of A. cinnamomea cells during fermentation. We observed some proteins such as phosphoribosylaminoimidazole-succinocarboxamide synthase, purine nucleotide biosynthesis-related protein, orotidine 5'-phosphate decarboxylase and elongation factor 2 showed up-regulation. Therefore, we inferred that the main metabolic pathway during exponential phase may emphasize the biosynthesis of nucleotides and proteins. The late-stationary phase signature revealed that up-regulation of pyruvate dehydrogenase complex E1-α subunit, squalene synthetase, redox-related and DNA repair-related proteins. The up-regulation of pyruvate dehydrogenase complex E1-α subunit and squalene synthetase may indicate that the biosynthesis of ergosterol and triterpenoids were mediated by switching the metabolic pool of acetyl-CoA, and the previous study indicated that in the late-stationary phase, A. cinnamomea cells have to face the reducing of medium nutritious and the increasing of the reactive oxygen species. The up-regulation of redox-related and DNA repair-related enzymes in the A. cinnamomea cells may reflect to the cells attempted to maintain metabolic redox homeostasis through regulating and coping with reactive oxygen species and nutritional stress. The foregoing results could understand the mechanism of metabolic regulation, and provide the applicability of proteome. Cordyceps sobolifera belongs to Cordyceps and is a rare and exotic medicinal fungus. It has been demonstrated that C. sobolifera has effects on protecting glomerular function from inflammation, decreasing the damage in renal interstitial cells and promoting the repair of renal tubule epithelial cells. This study was divided into two parts. The first part was using MTT assay to examine the effects of C. sobolifera extract (CSE), cyclosporin A (CSA) treated respectively and both two cotreated in NRK-52E rat kidney proximal renal tubule cell line for 24hrs. The data showed that 30 μM CSA -treated for 24hrs, the viability of NRK-52E cells were 46.6 % close to LC50, and when 250 μg/ml CSE and 30 μM CSA-cotreated, the viability of NRK-52E cells rose to 69.7 %. Results indicated that CSE could protect NRK-52E cells from the CSA-induced damage. The second part was to construct 2-DE patterns of intercellular proteins from NRK-52E cells for observing the changes of proteins and discussing the significance of these changes during the different treatments. We observed that some main proteins presented down-regulation in NRK-52E cells which were related to cell cycle, cell proliferation and differentiation while 30 μM CSA -treated for 24hrs, and the expression of these proteins increased in NRK-52E cells while 250 μg/ml CSE and 30 μM CSA-cotreated for 24hrs. The foregoing results may be helpful to understand the mechanism of CSE could protect NRK-52E cells from the CSA-induced damage.