Lu, Ying. / Thesis submitted in: March 2007. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 142-155). / Abstracts in English and Chinese. / THESIS COMMITTEE --- p.i / ACKNOWLEDGEMENTS --- p.iii / ABSTRACT(ENGLISH) --- p.iv / ABSTRACT(CHINESE) --- p.vi / TABLE OF CONTENT --- p.viii / LIST OF TABLES --- p.xii / LIST OF FIGURES --- p.xiv / ABBREVIATIONS --- p.xvii / Chapter CHAPTER 1: --- LITERATURE REVIEW --- p.1 / Chapter 1.1 --- Background of Sufu --- p.1 / Chapter 1.2 --- Classification of Sufu --- p.2 / Chapter 1.3 --- Production of Sufu --- p.3 / Chapter 1.3.1 --- Preparation of Tofu --- p.7 / Chapter 1.3.2 --- Preparation of Pehtze --- p.7 / Chapter 1.3.3 --- Salting and Brining --- p.8 / Chapter 1.3.4 --- Aging --- p.8 / Chapter 1.4 --- Biological and Chemical Changes during Sufu Production --- p.8 / Chapter 1.4.1 --- Microbial Changes during Sufu Production --- p.8 / Chapter 1.4.2 --- Proteolysis during Sufu Production --- p.9 / Chapter 1.4.3 --- Lipolysis during Sufu Production --- p.10 / Chapter 1.4.4 --- Flavor during Sufu Production --- p.10 / Chapter 1.5 --- Benefits of Sufu --- p.11 / Chapter 1.6 --- Existing Problems --- p.12 / Chapter 1.7 --- Exogenous Enzymes for Acceleration of Sufu Fermentation --- p.13 / Chapter 1.8 --- The Orthogonal Experimental Design --- p.14 / Chapter 1.9 --- Objective of This Study --- p.17 / Chapter CHAPTER 2: --- SURVEY ON PHYSICAL AND CHEMICAL PARAMETERS OF COMMERCIAL SUFU --- p.18 / Chapter 2.1 --- Introduction --- p.18 / Chapter 2.2 --- Materials and Methods --- p.19 / Chapter 2.2.1 --- Crude Protein Analysis --- p.19 / Chapter 2.2.2 --- Crude Fat Analysis --- p.19 / Chapter 2.2.3 --- Texture Profile Analysis (TPA) --- p.20 / Chapter 2.2.4 --- Free Amino Acid Analysis --- p.21 / Chapter 2.2.4.1 --- Chemicals and Standards --- p.23 / Chapter 2.2.4.2 --- Other Materials --- p.24 / Chapter 2.2.4.3 --- Additional Equipment --- p.24 / Chapter 2.2.4.4 --- Sample Pre-treament --- p.24 / Chapter 2.2.4.5 --- Preparing the Eluting Medium --- p.25 / Chapter 2.2.4.6 --- SPE and Derivatization --- p.25 / Chapter 2.2.4.7 --- GC-MS Conditions --- p.26 / Chapter 2.2.4.8 --- Calibration and Standard Curve Set Up --- p.26 / Chapter 2.2.4.9 --- Statistical Analysis --- p.27 / Chapter 2.2.5 --- Free Fatty Acid Analysis --- p.27 / Chapter 2.2.5.1 --- Chemicals and Standards --- p.27 / Chapter 2.2.5.2 --- Equipment --- p.28 / Chapter 2.2.5.3 --- Calibration and Standard Curve Set Up --- p.29 / Chapter 2.2.5.4 --- Free Fatty Acid Extraction --- p.29 / Chapter 2.2.5.5 --- GC-MS Conditions --- p.30 / Chapter 2.2.5.6 --- Statistical Analysis --- p.30 / Chapter 2.2.6 --- Sample Collection: Commercial Brands of Sufu --- p.31 / Chapter 2.3 --- Results --- p.32 / Chapter 2.3.1 --- Results of Crude Protein Contents in Commercial Sufus --- p.32 / Chapter 2.3.2 --- Results of Crude Fat Contents in Commercial Sufus --- p.33 / Chapter 2.3.3 --- Results of Texture Profile Analysis in Commercial Sufus --- p.34 / Chapter 2.3.4 --- Results of Free Amino Acids in Commercial Sufus --- p.37 / Chapter 2.3.5 --- Results of Free Fatty Acids in Commercial Sufus --- p.47 / Chapter 2.4 --- Discussion --- p.55 / Chapter CHAPTER 3: --- SHORTEN THE FERMENTATION TIME USING EXOGENOUS ENZYMES BY THE ORTHOGONAL EXPERIMENTAL DESIGN AND OPTIMIZE RESULTANT PROPERTIES --- p.58 / Chapter 3.1 --- Introduction --- p.58 / Chapter 3.2 --- Materials and Methods --- p.58 / Chapter 3.2.1 --- Laboratory-scale Sufu Production --- p.58 / Chapter 3.2.1.1 --- Preparation of Tofu --- p.58 / Chapter 3.2.1.2 --- Sub-culture Mold Strain --- p.59 / Chapter 3.2.1.3 --- Spore Suspension --- p.59 / Chapter 3.2.1.4 --- Preparation of Pehtze --- p.60 / Chapter 3.2.1.5 --- Inoculation of Tofu --- p.61 / Chapter 3.2.2 --- Brining and Aging with Addition of Enzyme Mixture --- p.62 / Chapter 3.2.3 --- Exogenous Enzymes of Food-grade --- p.62 / Chapter 3.2.3.1 --- Protamex --- p.63 / Chapter 3.2.3.2 --- Palatase --- p.64 / Chapter 3.2.3.3 --- Lipase --- p.64 / Chapter 3.2.3.4 --- Flavorzyme --- p.65 / Chapter 3.2.4 --- The Orthogonal Experimental Design --- p.65 / Chapter 3.2.4.1 --- Factors --- p.65 / Chapter 3.2.4.2 --- Statistical Analysis of Orthogonal Design L9 (34) --- p.66 / Chapter 3.2.5 --- "Crude Protein, Crude Fat and TPA Analysis" --- p.67 / Chapter 3.2.6 --- Free Amino Acid and Free Fatty Acid Analysis --- p.67 / Chapter 3.3 --- Results --- p.68 / Chapter 3.3.1 --- Orthogonal Results of Crude Protein Contents --- p.69 / Chapter 3.3.2 --- Orthogonal Results of Crude Fat Contents --- p.71 / Chapter 3.3.3 --- Orthogonal Results of Texture Profiles --- p.73 / Chapter 3.3.4 --- Orthogonal Results of Free Amino Acids --- p.80 / Chapter 3.3.5 --- Orthogonal Results of Free Fatty Acids --- p.108 / Chapter 3.4 --- Discussion --- p.121 / Chapter 3.4.1 --- Crude Protein of Enzyme Adding Sufu in Orthogonal Experiment --- p.121 / Chapter 3.4.2 --- Crude Fat of Enzyme Adding Sufu in Orthogonal Experiment --- p.122 / Chapter 3.4.3 --- Texture Profiles of Enzyme Adding Sufu in Orthogonal Experiment --- p.123 / Chapter 3.4.4 --- FAAs of Enzyme Adding Sufu in Orthogonal Experiment --- p.124 / Chapter 3.4.5 --- FFAs of Enzyme Adding Sufu in Orthogonal Experiment --- p.128 / Chapter CHAPTER 4: --- DISCUSSIONS AND CONCLUSION --- p.131 / REFERENCE --- p.142 / APPENDIX --- p.156

No description available.

Biologically active peptides are found in the amino acid sequence of bacterial, fungal, plant and animal proteins. They are among the most potent pharmacologically active agents. Examples are venom toxin (mellitin), hormones (oxytoxin), opioids (beta-endorphin) and enzyme inhibitors (hirudin). These bioactive peptides are released from proteins by enzymatic proteolysis by processes such as gastrointestinal digestion or food processing. / Soybeans were fermented with Bacillus subtilis ATCC 41332 and Rhizopus oligosporus NRRL 2710 to produce tempeh and natto, respectively. Samples were taken throughout the fermentation and analysed for biochemical changes. Protease activity and ammonia production detected in the early stage of the tempeh preparation suggested that protein was used as a carbon source during that period and contributed to a rise in pH. Bacillus subtilis did not produce ammonia and maintained a constant pH throughout the fermentation. The total peptides produced were at a maximum at the end of the fermentation cycles. Angiotensin-converting enzyme inhibitory activity increased throughout both fermentations. A method was developed to monitor the production of biogenic amines throughout the fermentation. The levels of biogenic amines increased dramatically as the fermentation proceeded until maturity was achieved. In the tempeh fermentation, the polyamines levels rose from the initial 19 ppm to final concentration of 862 ppm, with the largest increase in histamine (616 ppm) followed by putrescine (204 ppm). These compounds also contributed to the pH rise from 3.8 to 6.8 in 24 h. In the Bacillus fermentation, the total polyamines at the end of the fermentation was 110 ppm with the largest increase in putrescine, followed by cadaverine. / Soy hydrolysate and the soy fermented foods, natto and tempeh, were deglycosylated and treated with proteolytic enzymes (plasma proteases, kidney homogenate, pronase, pepsin, thermolysin, trypsin, chymotrypsin and proteinase K) to produce oligopeptides. Several peptides were isolated, purified and characterized. The peptides had a range of biological activities---angiotensin converting enzyme inhibitory, antithrombotic, surface tension, antibacterial, anti-oxidant and insulin-modulating activities. Three potent ACE inhibitors, three thrombin inhibitors, five peptides with surface-active properties and one peptide with antibacterial activity were identified. They were all derived from glycinin and were found in the plasma protease digest, the kidney homogenate digest and the pronase digest of fermented foods. Another sequence ELLVYLL possessed good surface active properties but its precursor could not be identified. However, it was analogous to a peptide produced by Bacillus subtilis , and was probably synthesized during fermentation. Peptide analogs were synthesized and evaluated. They showed similar activities. Other sequences of known inhibitors, TPKDFEEIPEE, FPRGGG and DFEEIPEEL, were found to be competitive substrates for ACE.

Chang Pui Sze. / Thesis submitted in: October 2004. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 204-227). / Abstracts in English and Chinese. / Acknowledgement --- p.i / Abstract (In English) --- p.ii / Abstract (In Chinese) --- p.iv / List of Tables --- p.vi / List of Figures --- p.x / Contents --- p.xii / Chapter 1. --- Introduction --- p.1 / Chapter 1.1 --- Historical Background of Sufu --- p.1 / Chapter 1.2 --- Nutritional Benefits of Sufu --- p.2 / Chapter 1.3 --- Production Steps --- p.2 / Chapter 1.3.1 --- Preparation of Tofu --- p.5 / Chapter 1.3.2 --- Preparation of Pehtze --- p.5 / Chapter 1.3.3 --- Salting or Brining --- p.8 / Chapter 1.3.4 --- Aging --- p.9 / Chapter 1.4 --- Local Varieties of Sufu --- p.9 / Chapter 1.5 --- Other Types of Sufu Fermentation --- p.10 / Chapter 1.6 --- Biochemical Changes during Sufu Production --- p.11 / Chapter 1.6.1 --- Protein Faction --- p.12 / Chapter 1.6.2 --- Lipid Fraction --- p.14 / Chapter 1.6.3 --- Carbohydrate fraction --- p.14 / Chapter 1.7 --- Sufu Flavor --- p.14 / Chapter 1.7.1 --- "Water-soluble Peptides, Free Amino Acids and Tasty Oligopeptides" --- p.14 / Chapter 1.7.2 --- Nucleotide Contents in the Taste of Sufu --- p.15 / Chapter 1.7.3 --- Effects of Ethanol on Flavor Development --- p.15 / Chapter 1.7.4 --- Volatile Components in Sufu --- p.16 / Chapter 1.8 --- Microbiological Safety of Sufu --- p.18 / Chapter 1.9 --- Existing Problems in Sufu Production --- p.19 / Chapter 1.10 --- Acceleration of Sufu Maturation by Adding Exogenous Lipase --- p.20 / Chapter 1.10.1 --- Proteases --- p.22 / Chapter 1.10.2 --- Problems with Proteases --- p.24 / Chapter 1.10.3 --- Lipases --- p.25 / Chapter 1.10.4 --- Problems with Lipases --- p.26 / Chapter 1.11 --- Replacement of Sodium Salt in Food with Alternative Salts --- p.29 / Chapter 1.12 --- Objectives --- p.33 / Chapter 2 --- Development of Volatile Compounds in Sufu --- p.35 / Chapter 2.1 --- Introduction --- p.35 / Chapter 2.2 --- Materials and Method --- p.36 / Chapter 2.2.1 --- Sufu Production --- p.36 / Chapter 2.2.1.1 --- Preparation of Tofu --- p.36 / Chapter 2.2.1.2 --- Inoculation of Tofu --- p.37 / Chapter 2.2.1.2.1 --- The Mold Strain --- p.37 / Chapter 2.2.1.2.2 --- Spore Suspension --- p.38 / Chapter 2.2.1.2.3 --- Spore Count in Spore Suspension --- p.38 / Chapter 2.2.1.3 --- Preparation of Pehtze --- p.39 / Chapter 2.2.1.4 --- Brining and Aging --- p.41 / Chapter 2.2.2 --- Sampling of Sufu --- p.42 / Chapter 2.2.3 --- Flavor Analysis --- p.42 / Chapter 2.2.3.1 --- Simultaneous Steam Distillation-Solvent Extraction (SDE) --- p.42 / Chapter 2.2.3.2 --- Gas chromatography-mass spectrometry (GC-MS) Conditions --- p.43 / Chapter 2.2.3.3 --- Compound Identification and Quantification --- p.44 / Chapter 2.3 --- Results --- p.45 / Chapter 2.3.1 --- Evolution of Volatiles During Sufu Aging --- p.45 / Chapter 2.3.1.1 --- Esters --- p.46 / Chapter 2.3.1.2 --- Alcohols --- p.51 / Chapter 2.3.1.3 --- Aldehydes --- p.55 / Chapter 2.3.1.4 --- Ketones --- p.55 / Chapter 2.3.1.5 --- Other Nitrogen-containing Compounds --- p.59 / Chapter 2.3.1.6 --- Sulfur (S)-containing and Oxygen (O)-containing Compounds --- p.51 / Chapter 2.3.1.7 --- Pyrazines --- p.61 / Chapter 2.3.1.8 --- Miscellaneous Compounds --- p.63 / Chapter 2.3.2 --- Change in the Concentrations of Sufu Odorous Compounds with Time --- p.65 / Chapter 2.4 --- Discussion --- p.67 / Chapter 2.4.1 --- Quantitatively Important Volatile Components of Sufu --- p.67 / Chapter 2.4.2 --- Esters --- p.59 / Chapter 2.4.3 --- Alcohols --- p.72 / Chapter 2.4.3.1 --- 1-Hexanol --- p.72 / Chapter 2.4.3.2 --- Phenol and 2-Methoxyphenol --- p.74 / Chapter 2.4.4 --- Aldehydes --- p.75 / Chapter 2.4.4.1 --- Hexanal --- p.75 / Chapter 2.4.4.2 --- "(E,E)-2,4-Heptadienal" --- p.77 / Chapter 2.4.4.3 --- (E)-2-Heptenal --- p.78 / Chapter 2.4.4.4 --- Benzeneacetaldehyde --- p.79 / Chapter 2.4.5 --- Ketones --- p.80 / Chapter 2.4.5.1 --- 3-Hydroxy-2-Butanone --- p.81 / Chapter 2.4.6 --- Sulfur-Containing Compounds --- p.82 / Chapter 2.4.6.1 --- 3-(Methylthio)propanal --- p.82 / Chapter 2.4.7 --- Pentylfuran --- p.84 / Chapter 2.4.8 --- Naphthalene --- p.86 / Chapter 2.4.9 --- Contaminants and artifacts generated by A-SDE --- p.87 / Chapter 2.5 --- Conclusion --- p.92 / Chapter 3 --- Acceleration of Sufu Production with Exogenous Lipase Effect on Flavor Development --- p.95 / Chapter 3.1 --- Introduction --- p.95 / Chapter 3.2 --- Materials and Method --- p.96 / Chapter 3.2.1 --- Sufu Production --- p.96 / Chapter 3.2.2 --- The Addition of Lipases --- p.96 / Chapter 3.2.3 --- Sampling of Sufu --- p.97 / Chapter 3.2.4 --- Flavor Analysis --- p.97 / Chapter 3.2.5 --- Statistical Analysis of Sufu Flavor Compounds --- p.98 / Chapter 3.2.6 --- Proximate Analysis --- p.98 / Chapter 3.2.7 --- Freeze-Drying --- p.99 / Chapter 3.2.8 --- Statistical Analysis of Sufu Proximate Contents --- p.99 / Chapter 3.2.9 --- Sensory Evaluation of Experimental Sufu --- p.100 / Chapter 3.3 --- Results --- p.102 / Chapter 3.3.1 --- Experiment I ´ؤ Adding 0.01% (w/w) Lipase from Porcine Pancreas and Candida rugosa --- p.102 / Chapter 3.3.1.1 --- Esters --- p.104 / Chapter 3.3.1.2 --- Alcohols --- p.108 / Chapter 3.3.1.3 --- Aldehydes --- p.110 / Chapter 3.3.1.4 --- 3-Hydroxy-2-Butanone --- p.113 / Chapter 3.3.1.5 --- 3-(Methylthio)propanal --- p.114 / Chapter 3.3.1.6 --- 2-Pentylfuran --- p.115 / Chapter 3.3.1.7 --- Naphthalene --- p.115 / Chapter 3.3.2 --- Experiment II - Adding 0.02% Lipase from Porcine Pancreas and Candida rugosa --- p.117 / Chapter 3.3.2.1 --- Esters --- p.118 / Chapter 3.3.2.2 --- Alcohols --- p.123 / Chapter 3.3.2.3 --- Aldehydes --- p.125 / Chapter 3.3.2.4 --- 3-Hydroxy-2 -Butanone --- p.129 / Chapter 3.3.2.5 --- 3-(Methylthio)propanal --- p.129 / Chapter 3.3.2.6 --- 2-Pentylfuran --- p.131 / Chapter 3.3.2.7 --- Naphthalene --- p.131 / Chapter 3.3.3 --- Sensory Evaluation of Lipase-treated Sufu --- p.132 / Chapter 3.3.4 --- Proximate Composition of Sufu at Different Ages from the 3 Treatments --- p.134 / Chapter 3.3.4.1 --- Addition of 0.01%(w/w) Lipase to Sufu Aging Solution --- p.134 / Chapter 3.3.4.1.1 --- Crude Protein --- p.134 / Chapter 3.3.4.1.2 --- Crude Lipid --- p.135 / Chapter 3.3.4.1.3 --- Moisture --- p.138 / Chapter 3.3.4.1.4 --- Ash --- p.138 / Chapter 3.3.4.2 --- Addition of 0.02% (w/w) Lipase to Sufu Aging Solution --- p.141 / Chapter 3.3.4.2.1 --- Crude Protein --- p.141 / Chapter 3.3.4.2.2 --- Crude Lipid --- p.141 / Chapter 3.3.4.2.3 --- Moisture --- p.144 / Chapter 3.3.4.2.4 --- Ash --- p.144 / Chapter 3.4 --- Discussion --- p.147 / Chapter 3.4.1 --- Adding 0.01 % Lipase from Porcine Pancreas and Candida rugosa --- p.147 / Chapter 3.4.1.1 --- Comparison of Total Odorous Content --- p.147 / Chapter 3.4.1.2 --- Sensory Evaluation of Experimental Sufu --- p.147 / Chapter 3.4.2 --- Adding 0.02% Lipase from Porcine Pancreas and Candida rugosai --- p.150 / Chapter 3.4.2.1 --- Comparison of Total Odorous Content --- p.150 / Chapter 3.4.2.2 --- Sensory Evaluation of Experimental Sufu --- p.151 / Chapter 3.4.3 --- Summary of Sensory and TOC Results of Lipase Experiments --- p.153 / Chapter 3.4.4 --- Impact of Lipase Addition on Different Odorous Volatile Compounds --- p.153 / Chapter 3.4.4.1 --- Esters --- p.154 / Chapter 3.4.4.2 --- Alcohols --- p.155 / Chapter 3.4.4.3 --- Aldehydes --- p.157 / Chapter 3.4.4.4 --- 3-Hydroxy-2-Butanone --- p.159 / Chapter 3.4.4.5 --- 3-(Methylthio)propanal --- p.160 / Chapter 3.4.4.6 --- 2-Pentylfuran and Naphthalene --- p.161 / Chapter 3.4.5 --- Effect of Aging on Chemical Composition of Sufu --- p.161 / Chapter 3.4.5.1 --- Crude Protein --- p.161 / Chapter 3.4.5.2 --- Crude Lipid --- p.162 / Chapter 3.4.5.3 --- Ash --- p.163 / Chapter 3.4.6 --- Effect of Lipase Addition on Chemical Composition of Sufu --- p.163 / Chapter 3.4.7 --- Effect of Lipase Addition on Free Fatty Acid (FFA) Profiles --- p.164 / Chapter 3.4.8 --- Generation of Different Classes of Esters from Animal and Fungal Lipases --- p.168 / Chapter 3.5 --- Conclusion --- p.171 / Chapter 4 --- "Partial Substitution of Sodium Chloride with Potassium Chloride in Sufu Aging Solution - Effect on Proteolysis, Bacterial Growth and Flavor" --- p.174 / Chapter 4.1 --- Introduction --- p.174 / Chapter 4.2 --- Materials and Method --- p.175 / Chapter 4.2.1 --- Sufu Production --- p.175 / Chapter 4.2.2 --- Partial Substitution of NaCl with KC1 --- p.176 / Chapter 4.2.3 --- Sampling of Sufu --- p.176 / Chapter 4.2.4 --- Bacterial Count --- p.176 / Chapter 4.2.5 --- Total and Amino Nitrogen Contents in Sufu --- p.177 / Chapter 4.2.6 --- Sensory Evaluation of Experimental Sufu --- p.177 / Chapter 4.3 --- Results --- p.179 / Chapter 4.3.1 --- Microbial Growth --- p.179 / Chapter 4.3.2 --- Proteolysis --- p.181 / Chapter 4.3.2.1 --- Total Nitrogen Content (TN) --- p.181 / Chapter 4.3.2.2 --- Amino Nitrogen Content (AN) --- p.184 / Chapter 4.3.3 --- Sensory Evalutaion of Control and KCl-Substituted Sufu --- p.186 / Chapter 4.4 --- Discussion --- p.187 / Chapter 4.4.1 --- Microbial Growth --- p.187 / Chapter 4.4.2 --- Proteolysis --- p.189 / Chapter 4.4.3 --- Sensory Tests --- p.194 / Chapter 4.5 --- Conclusion --- p.198 / Chapter 5 --- Overal Conclusion --- p.199 / References --- p.204 / Appendix I --- p.228 / Appendix II --- p.229

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

The conversion of soybeans to tempe is achieved through fermentation by Rhizopus. This fermentation process leads to hydrolysis of both proteins and lipids. The present work investigated certain biochemical changes which accompany the conversion of soybeans to tempe. The contents of non-protein nitrogen and free $ alpha$-amino nitrogen increased from 2.34 to 15.14%, and 2.03 to 5.22%, respectively after 48 h fermentation. SDS electrophoresis showed that a substantial quantity of the proteins in raw soybeans were hydrolysed by the Rhizopus to low molecular species (molecular weight $<$13,000 Daltons). Trypsin inhibitor activity found in tempe was lower than that of soybean and soaked soybean (an intermediate step in tempe preparation). The protein digestibilities of tempe and soaked soybean were higher than that of soybean. Reversed phase HPLC showed that the peptide separation profile of tempe was different from that of soybean and soaked soybean. The ESI/MS of the RP-HPLC fractions gave molecular weight of soybean peptides ranging from 1962 Da to 22,699 Da and tempe peptides ranging from 569 Da to 16,688 Da. The fatty acid compositions of tempe, soybean and soaked soybean were similar; relatively high levels of linoleic acid followed by oleic, linolenic and stearic acids were found. The acid values increased from 1.49 to 11.42 during the fermentation of soybeans. The total soluble carbohydrate contents of soybean, tempe and soaked soybean as well as the types and quantities of individual sugars were similar. The fermentation of soybean by Rhizopus had only a minor effect on the proximate composition of soybean; however, the soybean and fungal enzymes contributed primarily to changes in protein composition.

No description available.

Severe malnutrition in children is unacceptable. Rising malnutrition and household food insecurity are common. The problems of hunger and malnutrition in Nigeria are more severe than before. It has been estimated that the percentage of Nigerian households that are food insured was 40% in 1998 increasing from 18% in 1986. Malnutrition is widespread and its prevalence is high. ,The incidence of malnutrition has increased as a result of economic hardships facing the country. It is the children who suffer most. Their energy needs are never met and they remain hungry and wasted. UNICEF, 1998, published the number of malnourished under-five Nigerian children between 1990-1997 as 48% underweight, 9% wasted and 43% stunted. Families are unable to provide animal protein for the growing needs of the children. The use of soybean to augment meals lacking in animal protein is becoming popular. The Kersey Nutrition Rehabilitation Centre (KNRC) uses soybean as its mainstay in the rehabilitation of malnourished children. Reduction of soybean's high concentratioI1S of phytic acid will greatly enhance the crop's nutritional value, especially zinc and iron whose supplementation in the malnourished has greatly improved the management and achieved better weight gain. Up to now, the malnourished children in the world wait for deliverance from their burden. It is hoped that fermented soy supplements will reach out to these children more than ever.

Cheung Hiu-Ming. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 140-150). / Abstracts in English and Chinese. / ABSTRACT --- p.i / ACKNOWLEDGEMENTS --- p.vi / LIST OF FIGURES --- p.xiii / LIST OF TABLES --- p.xv / Chapter CHAPTER 1 --- LITERATURE REVIEW --- p.1 / Chapter 1.1 --- Introduction --- p.2 / Chapter 1.2 --- Soybeans --- p.3 / Chapter 1.2.1 --- Chemistry and nutritional value of soybeans --- p.3 / Chapter 1.2.2 --- Protein composition of soybeans --- p.4 / Chapter 1.2.3 --- Volatile compounds in soybeans --- p.4 / Chapter 1.3 --- Food fermentation --- p.5 / Chapter 1.4 --- Chaw tofu --- p.6 / Chapter 1.4.1 --- Preparation of tofu --- p.7 / Chapter 1.4.2 --- Preparation of chaw tofu --- p.7 / Chapter 1.4.3 --- Microorganisms involved in fermentation of chaw tofu --- p.8 / Chapter 1.4.4 --- Volatile components in chaw tofu --- p.11 / Chapter 1.4.5 --- Proteolytic activity of chaw tofu --- p.12 / Chapter 1.5 --- Stinky brine broth --- p.13 / Chapter 1.5.1 --- The pH value of stinky brine broth --- p.13 / Chapter 1.5.2 --- The salt percentage of stinky brine broth --- p.14 / Chapter 1.5.3 --- Volatile components of stinky brine broth --- p.14 / Chapter 1.5.4 --- Parameters affecting ammonia production of stinky brine --- p.15 / Chapter 1.6 --- Other fermented soy products --- p.16 / Chapter 1.6.1 --- Microorganisms involved in the fermentation --- p.16 / Chapter 1.6.1.1 --- Fermentation of soybean by bacteria --- p.17 / Chapter 1.6.1.1.1 --- Natto --- p.17 / Chapter 1.6.1.1.2 --- Kinema --- p.18 / Chapter 1.6.1.1.3 --- Soy daddawa --- p.19 / Chapter 1.6.1.1.4 --- Hawaijar --- p.20 / Chapter 1.6.1.1.5 --- Thua nao --- p.21 / Chapter 1.6.1.2 --- Fermentation of soybean by moulds --- p.21 / Chapter 1.6.1.2.1 --- Tempe --- p.21 / Chapter 1.6.1.2.2 --- Sufu --- p.22 / Chapter 1.6.1.2.3 --- Soy sauce --- p.22 / Chapter 1.6.1.2.4 --- Soy paste --- p.23 / Chapter 1.6.2 --- Formation of volatile compounds during Bacillus fermentation --- p.24 / Chapter 1.6.3 --- Biochemical changes during fermentation --- p.21 / Chapter 1.7 --- Methods of flavor analysis --- p.30 / Chapter 1.7.1 --- Headspace Analysis --- p.31 / Chapter 1.7.2 --- Aroma characterization --- p.32 / Chapter CHAPTER 2 --- IDENTIFICATION OF ODOROUS COMPOUNDS IN COMMERCIAL CHAW TOFU BASED ON ODOR ACTIVITY EVALUATION --- p.42 / Chapter 2.1 --- Introduction --- p.43 / Chapter 2.2 --- Materials & Methods --- p.46 / Chapter 2.2.1 --- Experimental samples --- p.46 / Chapter 2.2.2 --- Headspace-Gas Chromatography-Mass Spectrometry (GC-MS) --- p.46 / Chapter 2.2.3 --- Conditions of the Gas Chromatography-Mass Spectrometry (GC-MS) --- p.47 / Chapter 2.2.4 --- Compound identifications --- p.48 / Chapter 2.2.5 --- Quantification of compounds --- p.48 / Chapter 2.2.6 --- Statistical analysis --- p.48 / Chapter 2.2.7 --- Calculation of odor activity value (OAV) --- p.49 / Chapter 2.3 --- Results & Discussion --- p.50 / Chapter 2.3.1 --- Odor activity value (OAV) --- p.51 / Chapter 2.3.2 --- Volatile compounds in fresh samples --- p.51 / Chapter 2.3.2.1 --- Comparison of odorous compounds in fresh samples among different locations --- p.52 / Chapter 2.3.3 --- Volatile compounds in deep-fat fried samples --- p.53 / Chapter 2.3.3.1 --- Comparison of odorous compounds in deep-fat fried samples among different locations --- p.54 / Chapter 2.3.4 --- Comparison between fresh and deep-fat fried samples --- p.55 / Chapter 2.3.5 --- Odorous compounds of chaw tofu based on OAVs --- p.56 / Chapter 2.3.6 --- Possible ways for formation of odorous compounds --- p.58 / Chapter 2.3.6.1 --- Protein degradation --- p.58 / Chapter 2.3.6.2 --- Lipid degradation --- p.59 / Chapter 2.3.7 --- Comparison between volatile compounds in chaw tofu and others fermented soybean products --- p.60 / Chapter 2.4 --- Conclusion --- p.61 / Chapter CHAPTER 3 --- IDENTIFICATION OF ODOROUS COMPOUNDS IN COMMERCIAL CHAW TOFU BASED ON GAS CHROMATOGRAPHY-OLFACTOMETRY --- p.67 / Chapter 3.1 --- Introduction --- p.68 / Chapter 3.2 --- Materials & Methods --- p.71 / Chapter 3.2.1 --- Experimental samples --- p.71 / Chapter 3.2.2 --- Gas Chromatography-Mass Spectrometry-Flame Ionization Detection-Olfactometry (GC-MS-FID-O) --- p.71 / Chapter 3.2.3 --- Conditions of the Gas Chromatography-Mass Spectrometry --- p.72 / Chapter 3.2.4 --- Detection of odor active compounds --- p.73 / Chapter 3.2.5 --- Compound identifications --- p.73 / Chapter 3.3 --- Results & Discussion --- p.74 / Chapter 3.3.1 --- "Fecal, rancid and putrid odor" --- p.74 / Chapter 3.3.2 --- "Cabbages, sulfurous and meaty odor" --- p.76 / Chapter 3.3.3 --- Green odor --- p.77 / Chapter 3.3.4 --- Other odor contributing compounds --- p.77 / Chapter 3.3.5 --- Odor generate during deep-fat frying --- p.78 / Chapter 3.3.6 --- Comparison between GC-O and OAVs --- p.79 / Chapter 3.3.7 --- Comparison between volatile compounds in chaw tofu and others fermented soybean products --- p.80 / Chapter 3.4 --- Conclusion --- p.82 / Chapter CHAPTER 4 --- EVALUATION OF CHAW TOFU MODEL FERMENTATION BROTH --- p.86 / Chapter 4.1 --- Introduction --- p.87 / Chapter 4.2 --- Materials & Methods --- p.90 / Chapter 4.2.1 --- Model fermentation broth preparation --- p.90 / Chapter 4.2.2 --- Tofu sample preparation --- p.91 / Chapter 4.2.3 --- Gas Chromatography-Mass Spectrometry --- p.91 / Chapter 4.2.3.1 --- Conditions of Gas Chromatography-Mass Spectrometry (GC-MS) --- p.92 / Chapter 4.2.3.2 --- Compound identification --- p.93 / Chapter 4.2.3.3 --- Quantification of compounds --- p.93 / Chapter 4.2.4 --- Viable cell counts --- p.93 / Chapter 4.2.5 --- pH value and soluble content --- p.94 / Chapter 4.2.6 --- Proteolytic activity --- p.94 / Chapter 4.2.7 --- Statistical analysis --- p.95 / Chapter 4.3 --- Results & Discussion --- p.96 / Chapter 4.3.1 --- Headspaces analysis --- p.96 / Chapter 4.3.1.1 --- Changes in volatile composition in model fermentation broths --- p.97 / Chapter 4.3.1.2 --- Comparison of volatile compositions between the broths --- p.98 / Chapter 4.3.1.3 --- Comparison of volatile compositions among the three deep-fat fried fermented tofu with different broths --- p.101 / Chapter 4.3.1.4 --- Comparison of volatile compositions of deep fat fried fermented tofu with that of the commercial chaw tofu --- p.102 / Chapter 4.3.2 --- Liquid samples analysis --- p.104 / Chapter 4.3.2.1 --- "Changes in viable cell counts, pH values, protease activities and soluble solid contents within model fermentation broths during fermentation" --- p.106 / Chapter 4.3.2.2 --- Viable cell counts --- p.107 / Chapter 4.3.2.3 --- Soluble solid content --- p.108 / Chapter 4.3.2.4 --- Proteolytic activity --- p.106 / Chapter 4.3.2.5 --- pH value --- p.110 / Chapter 4.4 --- Conclusion --- p.112 / Chapter CHAPTER 5 --- GENERAL CONCLUSION --- p.127 / APPENDIX --- p.130 / IDENTIFICATION OF MICROORGANISMS PRESENTED IN THE MODEL CHAW TOFU FERMENTATION BROTHS BY MICROBIAL IDENTIFICATION SYSTEM (MIDI) --- p.130 / Materials & Methods --- p.130 / Model fermentation broth preparation --- p.130 / Viable cell counts --- p.131 / Microbial Identification System (MIDI) --- p.131 / Results --- p.133 / Suggestion on further investigation --- p.134 / REFERNECES --- p.141

腐乳是中國傳統豆類發酵製品，具有綿軟的口感和特殊的風味。其是豆腐通過真菌固態發酵，并加入鹽，米酒和香料等進行後期熟化而成的產品。本文的研究分為兩部份，第一部份對腐乳發酵過程中的毛胚，鹽胚，熟化第一天，熟化一個月以及熟化六個月的腐乳樣本進行採樣，并採用傳統微生物培養法和克隆文庫法對每個階段真菌和細菌的生態結構和動態變化進行研究。第二部份重點比較了四株腐乳產品中分離的微生物和購自台灣生物資源保存及研究中心的四株細菌的產揮發性含硫化合物能力，并挑選了最高產的一株微生物進行紫外誘變，最後獲得理想的突變株。本研究的結論如下： / 1. 真菌和細菌的總數均是在毛胚階段為最高，在進入熟化階段后開始下降。在傳統微生物培養方法下分別分離出了三株真菌和九株細菌，通過18S rDNA和16 rDNA測序，發現絲孢酵母屬（Trichosporon spp.）是真菌中的優勢菌種，蠟狀芽孢桿菌（Bacillus cereus）和解澱粉芽孢桿菌（Bacillus amyloliquefaciens）為細菌中的優勢菌種； / 2. 本研究建立了五個真菌18S rDNA克隆文庫和五個細菌16 rDNA克隆文庫用于研究真菌和細菌的生態結構和動態變化。通過聚合酶鏈式反應-限制性片段長度多態性（PCR-RFLP）的研究，分別在真菌和細菌克隆文庫中發現23和38種圖譜類型，并計算其相應比例。在進行真菌細菌測序之後，對優勢菌群進行了定性和定量分析； / 3. 在對比傳統微生物培養方法和克隆文庫技術的結果后發現，二者的結果存有差異，有些在克隆文庫中鑒定到的微生物在傳統培養方法中未能分離鑒定，而有些微生物則只能在傳統培養方法中被分離鑒定。因此，本研究中將這兩種方法結合有助於我們更為全面、客觀地研究腐乳發酵過程中真菌和細菌的生態結構和多樣性。 / 4. 對四株腐乳中分離純化的微生物和四株外來購入細菌的產揮發性含硫化合物能力進行比較，結果發現，從腐乳產品中分離純化的B-1菌株擁有最高的產揮發性含硫化合物能力，通過紫外誘變后，突變株#3在產揮發性含硫化合物以及L-蛋氨酸代謝酶活力都比初始菌株有了顯著的提升。B-1菌經測序比對后鑒定為絲孢酵母（Trichosporon sp.）。 / 本研究結果對于傳統腐乳發酵的有效控制和現代腐乳生產工藝的建立有一定指導意義，並且對於腐乳產品中的風味物質，特別是揮發性含硫化合物的產生和優化提供信息。 / Sufu (fermented soybean curd) is a soft creamy cheese-type product with a pronounced flavor and is made by fungal solid state fermentation of tofu (soybean curd) followed by aging in brine containing salt and alcohol. In first part of this research, the eco-structure and the dynamic changes of microbes during sufu production process (Pehtze, Salted pehtze, 0 Month sufu, 1 Month sufu and 6 Month sufu sample) were studied by combined microbiology techniques, including plate culture, 16S rDNA and 18S rDNA clone library and restriction fragment length polymorphism (RFLP) analysis. The second part of this research focus on the comparison of volatile sulfur-containing compounds (VSCs) production ability within isolated strains in sufu product and bacteria purchased commercially, the strain that possessed highest ability was selected and followed by a UV mutation experiment, finally obtained the desired mutant. The results of this research are as followed: / 1. The population of both fungi and bacteria were all at highest number in Pehtze stage and started to decrease in ripening stages. A combined total of three and nine living strains of fungi and bacteria were obtained from the plate culture, respectively. Through 18S rDNA and 16S rDNA sequencing, Trichosporon spp. was the dominant fungi and Bacillus cereus and Bacillus amyloliquefaciens were the dominant bacteria; / 2. Five 18S rDNA clone libraries and five 16S rDNA clone libraries from different stages of sufu production were constructed to analyze the structure and dynamic changes of fungi and bacteria. A total of 23 and 38 RFLP patterns were found, and the ratio of each pattern were calculated. After sequencing, qualitative and quantitative analysis on the dynamic changes of dominant strains was performed; / 3. After comparing the results of plate culture and clone library, it was found that there were some differences between the two. Some strains were only found in clone library while some only found in plate culture approach. Therefore, the combination of the two microbiology methods will help us to objectively and completely analyze the structure and dynamic changes of microbes in the sufu production process; / 4. The ability to produce VSCs within four strains (B-1, B-2, B-3 & B-4) isolated from a commercial sufu manufacturing process and four commercial strains (B. acetylicum, L. Lactics, S. thermophilus and L. Paracasei) were compared. Results showed that B-1 possessed both the highest VSCs production ability and L-methionine metabolism enzymatic activities among the eight strains. After UV light mutagenesis of B-1 strain, its mutant #3 significantly increased in DMDS and DMTS production and all four L-methionine-related enzymatic activities in reference to that of the starting strain (B-1). B-1 was identified as Trichosporon sp. by sequencing. / These results would make a profound significance on the control of traditional sufu production and the development of new technology for modern sufu manufacturing. They will also help to provide some important information of optimal production of VSCs in sufu ripening and the overall flavor in sufu product. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Huang, Ruolan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 106-117). / Abstracts also in Chinese. / Abstract --- p.i / 摘要 --- p.iii / Acknowledgement --- p.v / Table of contents --- p.vi / List of Figures --- p.x / List of Tables --- p.xiii / Chapter Chapter 1 --- : Introduction --- p.1 / Chapter 1.1 --- Sufu --- p.1 / Chapter 1.1.1 --- Classification --- p.1 / Chapter 1.1.1.1 --- Classified by processing technology --- p.1 / Chapter 1.1.1.2 --- Classified by color and flavor --- p.1 / Chapter 1.1.1.3 --- Other classifications --- p.2 / Chapter 1.1.2 --- Typical commercial manufacturing process --- p.2 / Chapter 1.1.2.1 --- Production process of naturally fermented sufu --- p.2 / Chapter 1.2.2.2 --- Production process of traditional mold-based sufu --- p.5 / Chapter 1.2.2.3 --- Production process of traditional bacteria-based sufu --- p.5 / Chapter 1.2.2.4 --- Acceleration of sufu ripening process --- p.6 / Chapter 1.1.3 --- Important ingredients in sufu production --- p.6 / Chapter 1.1.4 --- Flavor components in sufu --- p.7 / Chapter 1.1.4.1 --- Volatile flavor components --- p.7 / Chapter 1.1.4.2 --- Essential odor in sufu product --- p.8 / Chapter 1.1.4.3 --- Volatile sulfur compounds in sufu --- p.9 / Chapter 1.1.4.4 --- Using Head Space-Solid phase Microextraction (HS-SPME) to analyze the volatile sulfur components --- p.9 / Chapter 1.1.5 --- Relationship between microbes and sufu --- p.12 / Chapter 1.1.5.1 --- Microbes involved in fermentation process --- p.13 / Chapter 1.1.5.2 --- Microbial changes during the production of sufu --- p.14 / Chapter 1.1.6 --- Study on microbial ecology in food product --- p.15 / Chapter 1.1.6.1 --- PCR-based molecular techniques --- p.16 / Chapter 1.1.6.2 --- Non-PCR based molecular techniques --- p.16 / Chapter 1.1.6.3 --- The common techniques used in microbial ecology research --- p.17 / Chapter 1.1.6.4 --- Microbial ecology study by molecular biological techniques --- p.18 / Chapter 1.2 --- Objectives --- p.19 / Chapter Chapter 2 --- : Analysis of fungi diversity during sufu fermentation process --- p.21 / Chapter 2.1 --- Introduction --- p.21 / Chapter 2.2 --- Materials and methods --- p.21 / Chapter 2.2.1 --- Sample collection and preparation --- p.22 / Chapter 2.2.2 --- Plate count of fungi during sufu fermentation process --- p.22 / Chapter 2.2.3 --- Change of pH values and moisture content --- p.22 / Chapter 2.2.4 --- Total DNA extraction from fungi --- p.23 / Chapter 2.2.5 --- Preparation of competent cell --- p.23 / Chapter 2.2.6 --- 18S rDNA PCR amplification and construction of 18S rDNA clone library --- p.24 / Chapter 2.2.7 --- RFLP analysis of 18S rDNA clone library --- p.25 / Chapter 2.2.8 --- DNA sequencing for fungi identification --- p.26 / Chapter 2.2.9 --- Analysis of the diversity of 18S clone library --- p.26 / Chapter 2.2.10 --- Frequency percentage analysis --- p.27 / Chapter 2.2.11 --- Enzyme Solutions --- p.27 / Chapter 2.2.12 --- Determination of protease activity --- p.28 / Chapter 2.2.13 --- Determination of lipase activity --- p.29 / Chapter 2.2.11 --- Microtox test --- p.30 / Chapter 2.2.12 --- Statistical analysis --- p.30 / Chapter 2.3 --- Results and discussion --- p.30 / Chapter 2.3.1 --- Fungi growth on plate counting result --- p.30 / Chapter 2.3.2 --- Changes in pH and moisture content of sufu during production --- p.33 / Chapter 2.3.3 --- Construction and selection of 18S rDNA clone library --- p.35 / Chapter 2.3.4 --- Fungal diversity based on 18S rDNA clone library analysis --- p.38 / Chapter 2.3.5 --- Protease and lipase activities in Actinomucor elegans and Trichosporon japonicum --- p.45 / Chapter 2.3.5.1 --- Protease activity --- p.46 / Chapter 2.3.5.2 --- Lipase activity --- p.47 / Chapter 2.3.6 --- Toxicity of Actinomucor elegans and Trichosporon japonicum --- p.49 / Chapter 2.3.7 --- Analysis of fungi eco-structure and function during sufu fermentation process --- p.50 / Chapter 2.3.8 --- The influence of PCR bias and artifact --- p.53 / Chapter 2.2 --- Summary --- p.55 / Chapter Chapter 3 --- : Analysis of bacteria diversity during sufu fermentation process --- p.57 / Chapter 3.1 --- Introduction --- p.57 / Chapter 3.2 --- Materials and methods --- p.57 / Chapter 3.2.1 --- Sample collection and preparation --- p.57 / Chapter 3.2.2 --- Plate count of bacteria during sufu fermentation process --- p.57 / Chapter 3.2.3 --- Total DNA extraction from bacteria --- p.58 / Chapter 3.2.4 --- Preparation of competent cell --- p.58 / Chapter 3.2.5 --- 16S rDNA PCR amplification and construction of 16S rDNA clone library --- p.58 / Chapter 3.2.6 --- RFLP analysis of 16S rDNA clone library --- p.59 / Chapter 3.2.7 --- DNA sequencing for bacteria identification --- p.60 / Chapter 3.2.8 --- Analysis of the diversity of 16S rDNA clone library --- p.60 / Chapter 3.3 --- Results and discussion --- p.60 / Chapter 3.3.1 --- Bacteria growth on plate counting result --- p.60 / Chapter 3.3.2 --- Construction and selection of 16S rDNA clone library --- p.63 / Chapter 3.3.3 --- 16S rDNA clone library analysis of bacteria diversity --- p.65 / Chapter 3.3.4 --- Analysis of bacteria eco-structure and function during sufu fermentation process --- p.74 / Chapter 3.4 --- Summary --- p.77 / Chapter Chapter 4 --- : Screening the mutant possess higher capacity of forming volatile sulfur compounds (VSCs) from non-starter microbes of sufu product --- p.80 / Chapter 4.1 --- Introduction --- p.80 / Chapter 4.2 --- Materials and methods --- p.82 / Chapter 4.2.1 --- Strains and culture conditions --- p.82 / Chapter 4.2.2 --- Head space-solid phase microextraction (HS-SPME) analysis --- p.83 / Chapter 4.2.3 --- Gas Chromatography-Mass Spectrometry (GC-MS) analysis --- p.84 / Chapter 4.2.4 --- UV mutation --- p.85 / Chapter 4.2.5 --- Ellman’s method --- p.86 / Chapter 4.2.6 --- Preparation of cell-free extracts (CFE) for enzymatic assays --- p.86 / Chapter 4.2.7 --- Enzymatic assay --- p.86 / Chapter 4.2.7.1 --- L-methionine aminotransferase activity assay --- p.86 / Chapter 4.2.7.2 --- L-methionine demethiolase activity assay --- p.87 / Chapter 4.2.7.3 --- α-keto acid decarboxylase activity assay --- p.87 / Chapter 4.2.7.4 --- C-S lyase activity --- p.88 / Chapter 4.2.8 --- Statistical analysis --- p.88 / Chapter 4.3 --- Results and discussion --- p.89 / Chapter 4.3.1 --- Optimization of SPME extraction condition --- p.89 / Chapter 4.3.2 --- Selecting the start strain --- p.90 / Chapter 4.3.4.1 --- Comparison of VSCs production ability --- p.90 / Chapter 4.3.4.2 --- Comparison of enzymatic activity in L-methionine metabolism --- p.92 / Chapter 4.3.3 --- Optimization of UV exposure time --- p.95 / Chapter 4.3.4 --- Screening the mutants --- p.96 / Chapter 4.3.4.1 --- Comparison of VSCs production ability among the mutants --- p.96 / Chapter 4.3.4.2 --- Comparison of the L-methionine related enzymatic activities among the mutants --- p.99 / Chapter 4.3.4.3 --- Identified of strian B-1 --- p.101 / Chapter 4.4 --- Summary --- p.102 / Chapter Chapter 5 --- : General conclusions and future work --- p.103 / References --- p.106