腐乳是中國傳統的黃豆發酵類製品，口感柔滑，風味獨特，與西方的芝士相似。多個世紀以來一直是中國的特色配菜和開胃小吃。以往對腐乳的研究只限於使用水蒸氣蒸餾法，溶劑抽提法，超臨界萃取法以及頂空萃取法提取其揮發性化合物，但它們卻有準備時間長、可能產生製造物、較低靈敏度、使用有機溶劑或需要精密的設備等的缺點。而固相微萃取方法(SPME)是集取樣、提取、濃縮三個步驟為一的提取方法。這是一個簡單、快速、溫和而且有相對靈敏度較高的方法。因此，這項研究的主要目標是開發一個快速的頂空固相微萃取方法，並配合氣相色譜-質譜聯用方法來提取和鑒定巿面上白腐乳的揮發性物質，並研究白腐乳整個發酵週期的理化的變化。 / 第一部份的研究首先對固相微萃取塗層(萃取頭)，提取溫度，提取時間，鹽濃度和水含量等參數進行優化。優化條件的結果如下:(1)樣本對鹽溶液(NaCl溶液(25%飽和濃度))的比例為1:2(w/v)，(2)用Divinylbenzene/Carboxen/ Polydimethylsiloxane 塗層的萃取頭，(3)在55°C的水浴進行30分鐘提取。 / 第二部份的研究是用氣相色譜-質譜聯用方法分析利用以上的方法提取的三種商業白腐乳的揮發性化合物。結果在三個樣品中共檢測出131揮發性化合物。樣品A、B和C，分別檢測出112、112和118種成份，他們均屬於不同的官能團。三種腐乳共有76種相同的化合物。其中包括11種由Chung等人於2005年確定的重要香味化合物。而醇類和酯類化合物的含量最為豐富。另外，許多酯類，醛類和芳香烴化合物都是首次在腐乳中發現的。總括而言，該部份實驗成功開發了一種廉價且溫和的提取技術，讓我們可以簡單及快速地從檢定出腐乳中多種揮發性化合物。 / 研究的最後部份是針對商業白腐乳在發酵(腐乳坯期)和老化(後加入紅酒和鹽水)時，揮發性化合物的種類及濃度和理化參數的變化進行了研究。結果顯示，主要的化學成分的濃度是在發酵及熟成過程中増加，同時新的化合物亦不斷地形成。由腐乳坯階段至36小時、60天、120天和180天的發酵期，分別有37、58、69、83和86種化合物形成。其中酯類及吡嗪佔大多數，其濃度亦不斷地增加。而且，大部份的重要香味化合物的濃度亦顯著地增加。另外，許多在黃酒和芝麻油檢測出來的揮發物也可以從腐乳中找到的。理化分析的結果顯示，水分含量沒有顯著變化，但蛋白質的含量在熟化期的首四個月顯著下降。相反，灰含量則在四個月顯著地增加。此研究為腐乳發酵和熟化過程中的揮發性成分和化學成分的變化提供了更多的資料，然而要進一步了解腐乳發酵對味道和口感的影響則需要更多感官測試方面的研究。 / Sufu is a traditional Chinese fermented product with a soft creamy cheese-like texture and a unique flavour. It has been widely consumed in China as an appetizer for centuries. Previous investigations on its volatile compounds using simultaneous steam distillation and solvent extraction (SDE), supercritical fluid extraction (SFE) or headspace extraction may suffer from drawbacks such as artifact formation, the use of organic solvents and the need for sophisticated equipment. An alternative Solid-phase Microextraction (SPME) method integrates sampling, extraction, concentration into a single step method. However, this technique is highly sensitive to experimental conditions, careful optimization would be required to ensure a good extraction performance. / Our primary objective in this study was to develop a quick volatile profiling method using the Headspace - Solid-phase Microextraction - Gas Chromatography/Mass Spectrometry (HS-SPME-GC/MS) for subsequent studies on commercial products and their changes throughout the fermentation and ageing periods. Parameters including stationary phase (fiber coating), extraction temperatures, exposure times, concentration of salt and water content were optimized. / The optimal conditions found using the Divinylbenzene/Carboxen/ Polydimethylsiloxane (DVB/CAR/PDMS) fiber for sufu sample are (1) sample to 25% of (saturated) NaCl solution is 1:2 (w/v), (2) 30 min of extraction time at 55°C water bath. / The developed method was applied to study the volatile profile of three commercial brands of plain sufu. In total, 131 volatile compounds were detected in the headspace of the examined samples. Samples A, B and C have totals of 112, 112 and 118 compounds, respectively, and they belong to various chemical groups. Seventy-six compounds were found in common among the commercial samples. These included 11 out of the 14 aroma-impact compounds previously identified by Chung et al., 2005. Quantitatively, alcohols and esters were among the most abundant groups of compound found. Many esters, aldehydes and aromatic hydrocarbons compounds were first reported in this study. The results obtained by SPME were comparable to those obtained by SFE and SDE methods and at the same time it is cheaper and less labor intensive method in terms of the extraction and clean-up steps. In short, the developed HS-SPME method is an inexpensive, simple, rapid and mild extraction technique which allows the detection of a wide range of volatiles from sufu. / In the final part of the study, the changes of volatile profile and the physicochemical parameters of a commercially produced plain sufu were studied throughout its fermentation (pehtze period) and ageing (after wine & brine added) processes. The volatile profiles of the key ingredients, namely, yellow rice wine and sesame seed oil were also studied. In general, all the major chemical groups experienced an increase in concentration during ageing. Many new compounds were formed in the first few months of ageing. A total of 37, 58, 69, 83 and 86 compounds were identified in pehtze stage, days 0 (36 hour since bottling), 60, 120 and 180, respectively. Esters and pyrazines account for most of the quality difference. Their concentrations increased throughout the ageing period. Concentration for most of the aroma-impact compounds increased significantly throughout ageing. Many of the volatiles detected in the yellow rice wine and the sesame oil were found in common with the ageing or matured sufus (6th month). Proximate analysis showed that there were no significant changes in moisture content but a significant decline in both the lipid, protein contents were observed. On the other hand, the ash content was significantly increased in the first four months, but leveled off afterwards. While this study provides some more information to understand the changes in both volatile components and chemical composition during the fermentation and ageing processes, further studies will be needed to explain the flavour and textural changes. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Chiang, Tsz Kei Jackie. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 153-195). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts also in Chinese. / Thesis Committee / Acknowledgements / Abstract --- p.I / 摘要 --- p.IV / Table of Contents --- p.VI / List of Tables --- p.XI / List of Figures --- p.XII / List of Abbreviations --- p.XIV / Chapter Chapter 1 --- Literature Review --- p.1 / Chapter 1.1 --- Introduction --- p.1 / Chapter 1.2 --- Sufu --- p.3 / Chapter 1.2.1 --- History and Background of Sufu --- p.3 / Chapter 1.2.2 --- Sufu Classification --- p.5 / Chapter 1.2.2.1 --- White Sufu --- p.5 / Chapter 1.2.2.2 --- Red Sufu --- p.5 / Chapter 1.2.2.3 --- Grey Sufu --- p.6 / Chapter 1.2.2.4 --- Other Types --- p.6 / Chapter 1.2.3 --- Manufacturing of Sufu --- p.7 / Chapter 1.2.4 --- Microbes Used in Sufu Fermentation --- p.10 / Chapter 1.2.5 --- Biochemical Changes during Sufu Ageing --- p.10 / Chapter 1.2.5.1 --- Protein Faction --- p.11 / Chapter 1.2.5.2 --- Lipid Fraction --- p.12 / Chapter 1.2.5.3 --- Carbohydrate Fraction --- p.12 / Chapter 1.2.6 --- Flavour Origin of Sufu --- p.13 / Chapter 1.2.7 --- Volatile Components of Sufu --- p.14 / Chapter 1.2.7.1 --- Alcohols --- p.14 / Chapter 1.2.7.2 --- Esters --- p.15 / Chapter 1.2.7.3 --- Aldehydes --- p.16 / Chapter 1.2.7.4 --- Furans --- p.17 / Chapter 1.2.7.5 --- Ketones --- p.17 / Chapter 1.2.7.6 --- Sulphur-containing Compounds --- p.18 / Chapter 1.2.8 --- Aroma-impact Compounds --- p.20 / Chapter 1.2.8.1 --- Aroma-impact Compounds in Sufu --- p.23 / Chapter 1.3 --- Flavor Extraction Techniques --- p.23 / Chapter 1.3.1 --- Headspace Methods --- p.24 / Chapter 1.3.1.1 --- Static Headspace Sampling --- p.25 / Chapter 1.3.1.2 --- Dynamic Headspace Sampling --- p.25 / Chapter 1.3.2 --- Solvent Extraction --- p.26 / Chapter 1.3.3 --- Steam Distillation Techniques --- p.27 / Chapter 1.3.3.1 --- Simultaneous Steam Distillation and Solvent Extraction (SDE) --- p.27 / Chapter 1.3.3.2 --- High-vacuum Distillation Techniques --- p.28 / Chapter 1.3.4 --- Supercritical Fluid Extraction Methods (SFE) --- p.28 / Chapter 1.3.5 --- Solid-phase Extraction (SPE) --- p.30 / Chapter 1.3.6 --- Solid-phase Microextraction (SPME) --- p.31 / Chapter 1.4 --- Objectives --- p.35 / Chapter Chapter 2 --- Optimization of Headspace Solid-phase Microextraction Conditions for the Determination of Volatile Compounds in Sufu --- p.36 / Chapter 2.1 --- Introduction --- p.36 / Chapter 2.2 --- Materials and Methods --- p.40 / Chapter 2.2.1 --- Materials --- p.40 / Chapter 2.2.1.1 --- Commercial Plain Sufu --- p.40 / Chapter 2.2.1.2 --- SPME Accessories --- p.40 / Chapter 2.2.2 --- Sample Preparation --- p.41 / Chapter 2.2.3 --- Optimization of Sample Preparation Method for HS-SPME-GC/MS Analysis --- p.41 / Chapter 2.2.3.1 --- Effect of Ionic Strength (NaCl Concentration) --- p.42 / Chapter 2.2.3.2 --- Optimization of Water Content --- p.42 / Chapter 2.2.4 --- Optimization of the Extraction Conditions for HS-SPME- GC/MS Analysis --- p.42 / Chapter 2.2.4.1 --- HS-SPME Procedures --- p.43 / Chapter 2.2.5 --- Gas Chromatography/Mass Spectrometry (GC/ MS) Conditions --- p.43 / Chapter 2.2.6 --- Identification and Quantification of Selected Volatiles in Sufu --- p.44 / Chapter 2.3 --- Results and Discussions --- p.46 / Chapter 2.3.1 --- Method Development --- p.46 / Chapter 2.3.1.1 --- Choice of Fibers --- p.46 / Chapter 2.3.1.2 --- Effect of Time --- p.48 / Chapter 2.3.1.3 --- Effect of Temperature --- p.49 / Chapter 2.3.1.4 --- Effect of Ionic Strength (NaCl Concentration) --- p.51 / Chapter 2.3.1.5 --- Effect of Water Content --- p.52 / Chapter 2.3.2 --- Application of the HS-SPME-GC/MS Method for the Analysis of Sufu Volatiles --- p.61 / Chapter 2.3.2.1 --- Alcohols --- p.62 / Chapter 2.3.2.2 --- Aldehydes --- p.63 / Chapter 2.3.2.3 --- Aliphatic Hydrocarbons --- p.64 / Chapter 2.3.2.4 --- Aromatic Hydrocarbons --- p.65 / Chapter 2.3.2.5 --- Esters --- p.67 / Chapter 2.3.2.6 --- Furans --- p.69 / Chapter 2.3.2.7 --- Ketones --- p.70 / Chapter 2.3.2.8 --- Pyrazines --- p.71 / Chapter 2.3.2.9 --- Sulphur-containing Compounds --- p.73 / Chapter 2.3.3 --- Comparison of Different Extraction Techniques --- p.74 / Chapter 2.4 --- Conclusion --- p.86 / Chapter Chapter 3 --- Changes in Volatile Constituents and Physicochemical Characteristics of Commercial Plain Sufu throughout the Fermentation and Ageing Process --- p.87 / Chapter 3.1 --- Introduction --- p.87 / Chapter 3.2 --- Materials and Methods --- p.90 / Chapter 3.2.1 --- Sufu Sampling --- p.90 / Chapter 3.2.2 --- Flavour Analysis --- p.90 / Chapter 3.2.2.1 --- Sample Preparation --- p.90 / Chapter 3.2.2.2 --- Solid-phrase Microextraction (SPME) --- p.91 / Chapter 3.2.2.3 --- Gas Chromatography/ Mass Spectrometry (GC/ MS) Conditions --- p.92 / Chapter 3.2.2.4 --- Identification and Quantification of Selected Volatiles in Sufu --- p.93 / Chapter 3.2.3 --- Proximate Analysis --- p.93 / Chapter 3.2.4 --- Statistical Analysis --- p.94 / Chapter 3.3 --- Results and Discussions --- p.95 / Chapter 3.3.1 --- Overall Finding --- p.95 / Chapter 3.3.2 --- Flavour Analysis of Sufu at Different Fermentation and Ageing Stages --- p.97 / Chapter 3.3.2.1 --- Alcohols --- p.97 / Chapter 3.3.2.2 --- Aldehydes --- p.99 / Chapter 3.3.2.3 --- Esters --- p.101 / Chapter 3.3.2.4 --- Furans --- p.104 / Chapter 3.3.2.5 --- Ketones --- p.105 / Chapter 3.3.2.6 --- Pyrazines --- p.107 / Chapter 3.3.2.7 --- Sulphur-containing Compounds --- p.108 / Chapter 3.3.2.8 --- Miscellaneous Compounds --- p.110 / Chapter 3.3.2.9 --- Aroma-impact Compounds --- p.111 / Chapter 3.3.3 --- Potential Volatile Flavour Contributors from the Key Ingredients (Yellow Rice Wine and Sesame Seed Oil) --- p.112 / Chapter 3.3.3.1 --- Volatile Compounds in Yellow Rice Wine --- p.113 / Chapter 3.3.3.2 --- Volatile Compounds in Sesame Seed Oil --- p.114 / Chapter 3.3.4 --- Proximate Composition of Sufu at Different Fermentation and Ageing Stages --- p.115 / Chapter 3.3.5 --- Overall Discussion --- p.144 / Chapter 3.4 --- Conclusion --- p.146 / Chapter Chapter 4 --- OverallConclusion --- p.148 / Chapter 4.1 --- Conclusions and Significance of the Study --- p.148 / Chapter 4.2 --- Future Work --- p.151 / References --- p.153 / Chapter Appendix A --- p.196 / Chapter Appendix B --- p.199

No description available.

The potential of surfactants for improving the yields of alpha-amylase during the solid substrate fermentation (SSF) of potato starch using pure and mixed cultures was examined. The microorganisms used in this study were Aspergillus oryzae ATCC 1011, Bacillus subtilis ATCC 21556 and Bacillus subtilis ATCC 21332. The surfactants tested were Tween 20, Tween 80, SDS and surfactin. The fermentations were carried out in perforated trays after the addition of 10% (v/w) inoculum and with temperature and humidity controlled at 30°C and 90% RH respectively. Samples were taken and analyzed quantitatively for the production of alpha-amylase and biomass and qualitatively by scanning electron microscopy (SEM) using a JSM-840 A scanning microscope at 10 kV accelerating voltage. / It was possible to increase fungal alpha-amylase production by as much as 6 fold in the process with the addition of either synthetic surfactants or the biosurfactant surfactin. The bacterial alpha-amylase yields increased up to 11.5 fold in with the addition and/or the co-culture production of surfactants. The highest enzyme activity was found in the fermentation of a mixed culture of the two Bacillus strains with the addition of Tween 80. During the SSF with B. subtilis ATCC 21332 and ATCC 21556 as a mixed bacterial culture, there was also the production of surfactin in yields comparable to those obtained in a submerged fermentation. The biofilm formation as observed by SEM appeared to be associated with the presence of surfactants in the process and was not formed when no surfactants were present. The biofilm was observed as an entrapment of the bacteria in the substrate, resulting in improved access to the starch and higher production of alpha-amylase.

Aflatoxins are highly carcinogenic secondary metabolites that can contaminate approximately 25% of crops and that cause or exacerbate multiple adverse health conditions, especially in Sub-Saharan Africa and South and Southeast Asia. Regulation and decontamination of aflatoxins in high exposure areas is lacking. Biological detoxification methods are promising because they are assumed to be cheaper and more environmentally friendly compared to chemical alternatives. White-rot fungi produce non-specific enzymes that are known to degrade aflatoxin in in situ and ex situ experiments. The aims of this study were to (1) decontaminate aflatoxin-B-1-(AFB(1)) in naturally contaminated maize with the edible, white-rot fungus Pleurotus ostreatus (oyster mushroom) using a solid-state fermentation system that followed standard cultivation techniques, and to (2) and to assess the risk of mutagenicity in the resulting breakdown products and mushrooms. Vegetative growth and yield characteristics of P. ostreatus were not inhibited by the presence of-AFB(1).-AFB(1) was degraded by up to 94% by the Blue strain. No aflatoxin could be detected in P. ostreatus mushrooms produced from-AFB(1)-contaminated maize. Moreover, the mutagenicity of breakdown products from the maize substrate, and reversion of breakdown products to the parent compound, were minimal. These results suggest that P. ostreatus significantly degrades-AFB(1) in naturally contaminated maize under standard cultivation techniques to levels that are acceptable for some livestock fodder, and that using P. ostreatus to bioconvert crops into mushrooms can reduce-AFB(1)-related losses.

No description available.

Dependence on foreign oil remains a serious issue for the U.S. economy. Additionally, automobile emissions related to petroleum-based, fossil fuel has been cited as one source of environmental problems, such as global warming and reduced air quality. Using agricultural and forest biomass as a source for the biofuel ethanol industry, provides a partial solution by displacing some fossil fuels. However, the use of high cost enzymes as an input is a significant limitation for ethanol production.Economic analyses of cellulase enzyme production costs using solid state cultivation (SSC) are performed and compared to the traditional submerged fermentation (SmF) method. Results from this study indicate that the unit costs for the cellulase enzyme production are $15.67 per kilogram ($/kg) and $40.36/kg, for the SSC and SmF methods, respectively, while the market price for the cellulase enzyme is $36.00/kg. Profitability analysis and sensitivity analysis also provide positive results.Since these results indicate that the SSC method is economical, ethanol production costs may be reduced, with the potential to make ethanol a viable supplemental fuel source in light of current political, economic and environmental issues.

Orientador: Eleni Gomes / Banca: Adalbeto Pessoa Júnior / Banca: Maria de Lourdes Teixeira de Moraes Polizeli / Banca: Márcia Regina Brochetto Braga / Banca: Gustavo Orlando Bonilla Rodriguez / Resumo: Pectinases termoestáveis apresentam características interessantes do ponto de vista da sua aplicação industrial, como alta estabilidade ao pH e à temperatura. Além disso, o tipo de processo fermentativo pode influenciar a produção e propriedades físico-químicas destas enzimas. A produção de poligalacturonase (PG) pelo fungo Thermoascus aurantiacus foi realizada em fermentação submersa (FSM) e em estado sólido (FES), usando substratos contendo pectina comercial ou subprodutos agro- industriais como fonte de carbono. A PG bruta obtida em FES apresentou atividade ótima a 65ºC e pH 5,0, com estabilidade na faixa de pH entre 4,0 e 5,0 e entre 7,5 e 8,5 e manteve 85% da atividade original quando incubada a 60ºC, por 1 hora. Em FSM, o melhor meio de cultivo foi a água amarela, com pH inicial de 5,5, após 5 dias de cultivo a 45ºC. A enzima em sua forma bruta apresentou temperatura ótima de 60ºC e pH ótimo de 5,0, maior estabilidade em pH ácido (3,0 a 4,5) e menor termoestabilidade, quando comparada com a obtida em FES, mantendo apenas 13% da atividade original quando incubada a 60ºC, por 1 hora. As enzimas foram purificadas utilizando-se cromatografias de filtração em gel e troca iônica. A PG purificada proveniente da FSM apresentou pH e temperatura ótimos de 5,5 e 60-65ºC, estabilidade em pH 5,0-5,5 e manteve, após 1 hora de incubação, 100% da atividade original até 50ºC. Resultados similares foram obtidos para a PG proveniente da FES. A PG de FES apresentou massa molar de 29,3 kDa, Km de 1,58 mg/mL e Vmáx de 1553,1 ? mol/min/mg, enquanto que a da FSM apresentou massa molar de 30,1 kDa, km de 1,46 mg/mL e Vmáx de 2433,3 ? mol/min/mg. Íons como Fe+3, Ca+2, e K+ praticamente não afetaram a atividade da enzima... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Thermostable pectinases present important characteristics under the view of their industrial application, as their high stability to pH and temperature. Besides, the type of fermentative process used can affect the ir production and physical-chemical properties. The polygalacturonase (PG) production by the thermophilic fungus Thermoascus aurantiacus was carried out by submerged fermentation (SMF) and solid state fermentation (SSF) using substrates containing commercial pectin or agro- industrial residues as carbon sources. The crude PG from SSF presented optimum activity at 65ºC and pH 5.0, with stability at pH 4.0-5.0 and 7.5-8.5 and maintained 85% of its original activity at 60º C for 1 hour. In SMF the best cultivation medium was the liquid waste from juice extraction, with initial pH of 5.5, after 5 days of cultivation at 45ºC. The crude enzyme showed an optimum activity at 60ºC and pH 5.0, higher stability in acid ic pH (3.0 to 4.5) and was less thermostable when compared to that obtained in SSF, wich maintained only 13% of its original activity at 60ºC, for 1 hour. Purification of enzymes was carried out using filtration and ion-exchange chromatographies. The purified PG, from SMF, showed optimum pH and temperature of 5.5 and 60-65ºC, stability at pH 5.0-5.5 and preserved, after 1 hour incubation, 100% of its original activity at 50ºC. Similar results were obtained to PG from SSF. The PG obtained by SSF presented molar mass of 29.3 kDa, Km of 1.58 mg/ml and Vmáx of 1553.1 ? mol/min/mg, while that the enzyme from SMF presented molar mass of 30.1 kDa, km of 1.46 mg/ml and Vmáx of 2433.3 ? mol/min/mg. Ions such as Fe3+, Ca2+ and K+ practically did not affect the enzyme activity, while Mg2+, Mn2+ and Zn2+ decreased 7%, 75% and 50%... (Complete abstract, click electronic address below) / Doutor

M. Tech. (Department of Biotechnology, Faculty of Applied and Computer Sciences), Vaal University of Technology / Solid-state fermentation (SFF) has emerged as a good method for the production of microbial enzymes such as inulinases. The use of low-cost agricultural plants and agro-industrial residues as substrates in SSF processes provides a value adding alternative to these otherwise under/or un-utilised vegetation. Production of inulinases, using various inulin-containing plant materials as carbon sources was studied using pure and mixed cultures of yeast strains. All substrates resulted in different levels of enzyme activity. A mixed culture of Kluyveromyces marxianus and Saccharomyces cerevisiae produced an extracellular exoinulinase when grown on different types of inulin-containing plant materials. Initial inulinase production was achieved as follows: 10 IU/gds (garlic cloves), 15 IU/gds (parsnips), 10 IU/gds (wheat bran) and 7 IU/gds (amadumbe) by K. marxianus and S. cerevisiae in a mixed culture. The production of inulinases by a mixed culture of K. marxianus and S. cerevisiae under SSF was further optimized by investigating initial moisture content, temperature, carbon source, nitrogen source, inoculum volume and inoculum ratio. The highest inulinase activity attained was in garlic cloves (85 IU/gds), followed by parsnips (65 IU/gds), wheat bran (37 IU/gds) and amadumbe (25 U/gds). The activities yielded 5.6 fold higher inulinase than in preliminary studies. The optimum pH and temperature of the crude enzyme were 5.0 and 50 oC, respectively. The pH and temperature stability of the enzyme was steady for 1 hour retaining about 64% activity. The average inulinase/invertase activity (I/S) ratio of 1.0 by crude inulinases was also observed after 48 hours. The crude extracellular enzyme extracts from the garlic cloves, parsnips, amadumbe and wheat bran were partially purified by ammonium sulphate precipitation and showed a specific activity of 9.03 U/mg, 0.08 U/mg, 4.12 U/mg and 0.133 U/mg respectively. The Km and Vmax values of the inulinase were 21.95 mM and 2.09 μM/min; 19.79 mM and 1.38 μM/min; 31.59 mM and 0.51 μM/min; and 25.74 mM and 0.23 μM/min, respectively. All extracts demonstrated potential for large-.scale production of inulinase and fructose syrup.

Thesis (MSc (Microbiology))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: Long chain omega-3 fatty acids such as eicosapentaenoic acid (EPA) are essential for the regulation of critical biological functions in humans and other mammals. Fish oil as the main dietary source of EPA holds several disadvantages and alternative sources and production processes such as microbial fermentation are increasingly being investigated. Therefore the aim of the first part of this study was to evaluate brewers’ spent grain (BSG) as substrate for the production of EPA by solid state fermentation with 29 fungal strains representing different Mortierella species. The effect of a 10% (w/w) linseed oil (LSO) supplement on EPA production was also studied. Consequently, fungal inoculated BSG was incubated at 22oC for three days to obtain optimal fungal growth, before the incubation temperature was lowered to 16oC for the following eight days. Cultures were then harvested and dried, followed by lipid extraction and analyses using gas chromatography. All the strains were found to produce EPA on BSG, while addition of the LSO improved the EPA yield of most strains. The strains which produced the highest levels of EPA on BSG supplemented with LSO were Mortierella antarctica Mo 67 and Mortierella epicladia Mo 101, which respectively produced 2.8 mg and 2.5 mg EPA per g of BSG. During the second part of the study eight Mortierella strains were used to study EPA production via solid state fermentation of sunflower press cake (SPC). Similar culture conditions and analytical methods were used as in the first part of the study. The effect of supplementing the SPC substrate with 10% (w/w) LSO was studied with regard to the supplement’s impact on EPA production and on the highly unsaturated fatty acid (HUFA) profile of the fermented substrate. Addition of LSO improved EPA yield of most strains on SPC, leading to a reduction in the average arachidonic acid (ARA):EPA ratio from 50.68 to 3.66. The ratio of HUFA to saturated and monoenoic fatty acids, was increased significantly (t=5.75, p=0.05) by the addition of LSO, with higher desaturation levels among the 20-carbon fatty acids. Addition of LSO also had a positive effect (r = 0.9291, p = 0.001) on the relative amount of long chain fatty acids (C≥20) produced. The strains which produced the highest levels of EPA on SPC supplemented with LSO were Mortierella alpina Mo 46 and Mortierella basiparvispora Mo 88, which produced 6.4 mg and 5.8 mg EPA per g of sunflower press cake, respectively. Fungi belonging to the genus Mortierella successfully converted LSO supplemented agro-processing wastes, such as BSG and SPC, to materials containing EPA, thereby adding value to these substrates. These EPA-enriched waste substrates could eventually find applications as animal or fish feed or as a source of EPA and other HUFA for the growing omega-3 market in the neutraceutical and therapeutics industry. / AFRIKAANSE OPSOMMING: Langketting omega-3 vetsure soos eikosapentaenoë suur (EPS) is noodsaaklik vir die regulasie van kritiese biologiese funksies in mense en ander soogdiere. Visolie, die mees belangrike EPS-bron in die dieet, hou verskeie nadele in en alternatiewe bronne sowel as produksie-prosesse, soos mikrobiologiese fermentasie, word dus toenemend ondersoek. Die doel van die eerste gedeelte van hierdie studie was dus om gebruikte brouersgraan (GBG) te ëvalueer as ‘n substraat vir die produksie van EPS deur soliede staat fermentasie met 29 fungus isolate wat verskillende Mortierella spesies verteenwoordig. Die uitwerking van byvoeging van 10% (m/m) lynsaadolie (LSO) op EPS-produksie is ook bepaal. Gevolglik is fungus-geïnokuleerde GBG vir drie dae by 22oC geïnkubeer om optimale fungusgroei te verkry, waarna die inkubasie temperatuur verlaag is na 16oC vir die volgende agt dae. Kulture is hierna ge-oes en gedroog, gevolg deur lipied ekstraksie en analise met behulp van gaschromatografie. Al die isolate het EPS geproduseer op die GBG substraat, terwyl byvoeging van LSO die EPS-opbrengs van die meeste isolate verbeter het. Die isolate wat die hoogste vlakke van EPS op GBG wat met LSO verryk is, geproduseer het, was Mortierella antarctica Mo 67 en Mortierella epicladia Mo 101, wat onderskeidelik 2.8 mg en 2.5 mg EPS per g GBG geproduseer het. Tydens die tweede gedeelte van die studie is agt Mortierella isolate gebruik om die produksie van EPS deur soliede staat fermentasie van sonneblom perskoek (SPK) te ondersoek. Kultuurtoestande en analitiese metodes soortgelyk aan die eerste gedeelte van die studie is gebruik. Die uitwerking van byvoeging van 10% LSO tot die SPK substraat is ondersoek met betrekking tot die impak van die byvoeging op EPS produksie asook op die profiel van hoogs onversadigde vetsure (HOVS) van die gefermenteerde substraat. Die byvoeging van LSO tot SPK het die EPS opbrengs van meeste isolate verbeter en het tot ‘n verlaging in die gemiddelde arachidoonsuur (ARS):EPS verhouding vanaf 50.69 tot 3.66 gelei. Die verhouding van HOVS tot versadigde en mono-onversadigde vetsure, is betekenisvol (t=5.75, p=0.05) verhoog deur die byvoeging van LSO, met hoër vlakke van onversadigheid onder die 20-koolstof vetsure. Byvoeging van LSO het ook ‘n positiewe uitwerking (r = 0.9291, p = 0.001) op die relatiewe aantal langketting vetsure (C≥20) gehad. Die isolate wat die hoogste vlakke van EPS geproduseer het op LSO-verrykte SPK, was Mortierella alpina Mo 46 en Mortierella basiparvispora Mo 88, wat onderskeidelik 6.4 mg en 5.8 mg EPS per g SPK geproduseer het. Fungi wat aan die genus Mortierella behoort, het LSO-verrykte agroprosesserings afvalprodukte, soos GBG en SPK, suksesvol omgeskakel na materiale wat EPS bevat, en sodoende waarde toegevoeg aan hierdie substrate. Die EPS-verrykte afvalsubstrate kan uiteindelik toepassings vind as diere- of visvoer of as bron van EPS of ander HOVS vir die groeiende omega-3 mark in die neutraseutiese en terapeutiese industrie.

Na produção do etanol 2G, as celulases representam um componente importante do custo. Com base em informações da literatura e resultados obtidos no Laboratório de Engenharia Bioquímica da Escola Politécnica da USP, foi feito um estudo do custo de produção das celulases via fermentação em estado sólido (FES) em escala industrial. Foram consideradas as seguintes variáveis no processo de produção das celulases, denominadas cenários de produção: concentração das celulases no meio de cultura de 1 a 150 FPU/gms; produtividade em celulases em 0,11 e 0,45FPU/gms.h; atividade de celulase na hidrólise de bagaço de cana-de-açúcar entre 7 e 30 FPU/g de substrato seco; massa de bagaço de cana a ser hidrolisada entre 5 e 30% do bagaço gerado numa usina sucroalcooleira de referência (1.000.000 ton. de cana-de-açúcar /ano); custo dos substratos para a FES variando entre US$6,00 a US$12,00 por tonelada para o bagaço de cana-de-açúcar (80% da massa do meio) e US$80,00 a US$110,00 por tonelada para o farelo de trigo (20% da massa do meio); capacidade volumétrica dos reatores de FES variando entre 5 e 50 m3. Os impactos das variáveis consideradas foram: concentração de celulases no meio de cultura, acima de 45FPU/gms causam reduções menores que 5% no custo de produção; produtividades iguais em celulases (0,45FPU/gms.h), apresenta um processo 11% menos custoso para aquela com maior produção de enzimas, em relação ao tempo de fermentação; destinação de até 10% do bagaço gerado pela usina, reduz em 7% o custo de produção das celulases e a dosagem de celulase não causa redução significativa nos custos de produção, assim como o custo das matérias-primas; o aumento da capacidade dos reatores de FES causam redução de até 47% no custo de produção das celulases. A análise de regressão linear das variáveis apresentadas indica qual o impacto percentual dobre o custo de produção das celulases: quantidade de bagaço de cana-de-açúcar destinado à hidrólise (0,6%), atividade enzimática para a reação de hidrólise (0,3%), produção de celulase (73,4%), volume dos reatores de inóculo (0,5%), volume dos reatores de FES (2,7%), tempo de crescimento do inóculo (0,1%), tempo de fermentação (3,7%), custo do bagaço de cana-de-açúcar (0,9%), custo do farelo de trigo (0,6%) e outras variáveis não analisadas (17,2%). Conclui-se que, com os dados atuais sobre FES, a probabilidade de se obter um processo em escala industrial com custo de produção de celulase menor que US$0,50/100.000FPU é de 15%. Desenvolvimento de culturas microbianas mais produtivas em celulases e tecnologias mais avançadas de reatores de grande escala, são necessárias à viabilidade da produção das celulases via FES para aplicação na hidrólise de celulose. / In ethanol 2G production, cellulases are an important component of the cost. Based on information from literature and results obtained in the Labotatório de Engenharia Bioquímica da Escola Politécnica da USP, a study was made of the production cost of cellulases via solid state fermentation (SSF) on industrial scale. The following variables were considered in the production of cellulases, called \"production scenarios\": the concentration of cellulase in the culture medium from 1 to 150 FPU / gms; productivity cellulases in 0.11 and 0.45 FPU / gms.h; cellulase activity in the hydrolysis of bagasse from sugar cane between 7 and 30 FPU / g substrate dry mass; cane bagasse hydrolyzed to be between 5 and 30% of sugarcane bagasse generated in a reference plant (1,000,000 ton of cane sugar per year) and the cost of substrates for SSF ranging from $ 6.00 to $ 12.00 per ton for the sugarcane bagasse (80% by weight of medium) and $ 80, 00 to $ 110.00 per ton for wheat bran (20% of the mass medium); volumetric capacity of the reactors of FES ranged between 5 and 50 m3. The impacts of the variables considered were: concentration of cellulase in the culture medium above 45FPU/gms cause reductions of less than 5% of the cost of production; for equal productivities of cellulose (0.45 FPU / gms.h) shows a process 11% less costly when the production of enzymes is higher in relation to fermentation time; using up to 10% of sugarcane bagasse generated by the reference plant, reduces by 7% the cost of production of cellulases and cellulase dosage does not cause significant reduction in production costs as well as the cost of raw materials; increasing the capacity of the reactors of FES cause a reduction of up to 47% on cost of production of cellulases. The linear regression analysis of the variables presented indicates what percentage impact the production cost of cellulases: The amount of bagasse cane sugar for the hydrolysis (0.6%), enzymatic activity for the hydrolysis reaction (0, 3%), production of cellulase (73.4%), the reactor volume of inoculum (0.5%), volume of the reactors of FES (2.7%), time of growth of the inoculum (0.1%) fermentation time (3.7%), cost of sugarcane bagasse (0.9%), cost of wheat bran (0.6%) and other variables not analyzed (17.2%). It was concluded that with current data on FES, the probability of obtaining a process on an industrial scale with production cost of cellulase less than $ 0.50/100.000FPU is 15%. Development of microbial cultures more productive in cellulases and more advanced technologies for large-scale reactors are necessary for the viability of the production of cellulases for FES for application in the hydrolysis of cellulose.