Microbial ecology and the relationship between volatile sulfur-containing compound (VSCs) production and bacteria during sufu fermentation.

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.iiiAcknowledgement --- p.vTable of contents --- p.viList of Figures --- p.xList of Tables --- p.xiiiChapter Chapter 1 --- : Introduction --- p.1Chapter 1.1 --- Sufu --- p.1Chapter 1.1.1 --- Classification --- p.1Chapter 1.1.1.1 --- Classified by processing technology --- p.1Chapter 1.1.1.2 --- Classified by color and flavor --- p.1Chapter 1.1.1.3 --- Other classifications --- p.2Chapter 1.1.2 --- Typical commercial manufacturing process --- p.2Chapter 1.1.2.1 --- Production process of naturally fermented sufu --- p.2Chapter 1.2.2.2 --- Production process of traditional mold-based sufu --- p.5Chapter 1.2.2.3 --- Production process of traditional bacteria-based sufu --- p.5Chapter 1.2.2.4 --- Acceleration of sufu ripening process --- p.6Chapter 1.1.3 --- Important ingredients in sufu production --- p.6Chapter 1.1.4 --- Flavor components in sufu --- p.7Chapter 1.1.4.1 --- Volatile flavor components --- p.7Chapter 1.1.4.2 --- Essential odor in sufu product --- p.8Chapter 1.1.4.3 --- Volatile sulfur compounds in sufu --- p.9Chapter 1.1.4.4 --- Using Head Space-Solid phase Microextraction (HS-SPME) to analyze the volatile sulfur components --- p.9Chapter 1.1.5 --- Relationship between microbes and sufu --- p.12Chapter 1.1.5.1 --- Microbes involved in fermentation process --- p.13Chapter 1.1.5.2 --- Microbial changes during the production of sufu --- p.14Chapter 1.1.6 --- Study on microbial ecology in food product --- p.15Chapter 1.1.6.1 --- PCR-based molecular techniques --- p.16Chapter 1.1.6.2 --- Non-PCR based molecular techniques --- p.16Chapter 1.1.6.3 --- The common techniques used in microbial ecology research --- p.17Chapter 1.1.6.4 --- Microbial ecology study by molecular biological techniques --- p.18Chapter 1.2 --- Objectives --- p.19Chapter Chapter 2 --- : Analysis of fungi diversity during sufu fermentation process --- p.21Chapter 2.1 --- Introduction --- p.21Chapter 2.2 --- Materials and methods --- p.21Chapter 2.2.1 --- Sample collection and preparation --- p.22Chapter 2.2.2 --- Plate count of fungi during sufu fermentation process --- p.22Chapter 2.2.3 --- Change of pH values and moisture content --- p.22Chapter 2.2.4 --- Total DNA extraction from fungi --- p.23Chapter 2.2.5 --- Preparation of competent cell --- p.23Chapter 2.2.6 --- 18S rDNA PCR amplification and construction of 18S rDNA clone library --- p.24Chapter 2.2.7 --- RFLP analysis of 18S rDNA clone library --- p.25Chapter 2.2.8 --- DNA sequencing for fungi identification --- p.26Chapter 2.2.9 --- Analysis of the diversity of 18S clone library --- p.26Chapter 2.2.10 --- Frequency percentage analysis --- p.27Chapter 2.2.11 --- Enzyme Solutions --- p.27Chapter 2.2.12 --- Determination of protease activity --- p.28Chapter 2.2.13 --- Determination of lipase activity --- p.29Chapter 2.2.11 --- Microtox test --- p.30Chapter 2.2.12 --- Statistical analysis --- p.30Chapter 2.3 --- Results and discussion --- p.30Chapter 2.3.1 --- Fungi growth on plate counting result --- p.30Chapter 2.3.2 --- Changes in pH and moisture content of sufu during production --- p.33Chapter 2.3.3 --- Construction and selection of 18S rDNA clone library --- p.35Chapter 2.3.4 --- Fungal diversity based on 18S rDNA clone library analysis --- p.38Chapter 2.3.5 --- Protease and lipase activities in Actinomucor elegans and Trichosporon japonicum --- p.45Chapter 2.3.5.1 --- Protease activity --- p.46Chapter 2.3.5.2 --- Lipase activity --- p.47Chapter 2.3.6 --- Toxicity of Actinomucor elegans and Trichosporon japonicum --- p.49Chapter 2.3.7 --- Analysis of fungi eco-structure and function during sufu fermentation process --- p.50Chapter 2.3.8 --- The influence of PCR bias and artifact --- p.53Chapter 2.2 --- Summary --- p.55Chapter Chapter 3 --- : Analysis of bacteria diversity during sufu fermentation process --- p.57Chapter 3.1 --- Introduction --- p.57Chapter 3.2 --- Materials and methods --- p.57Chapter 3.2.1 --- Sample collection and preparation --- p.57Chapter 3.2.2 --- Plate count of bacteria during sufu fermentation process --- p.57Chapter 3.2.3 --- Total DNA extraction from bacteria --- p.58Chapter 3.2.4 --- Preparation of competent cell --- p.58Chapter 3.2.5 --- 16S rDNA PCR amplification and construction of 16S rDNA clone library --- p.58Chapter 3.2.6 --- RFLP analysis of 16S rDNA clone library --- p.59Chapter 3.2.7 --- DNA sequencing for bacteria identification --- p.60Chapter 3.2.8 --- Analysis of the diversity of 16S rDNA clone library --- p.60Chapter 3.3 --- Results and discussion --- p.60Chapter 3.3.1 --- Bacteria growth on plate counting result --- p.60Chapter 3.3.2 --- Construction and selection of 16S rDNA clone library --- p.63Chapter 3.3.3 --- 16S rDNA clone library analysis of bacteria diversity --- p.65Chapter 3.3.4 --- Analysis of bacteria eco-structure and function during sufu fermentation process --- p.74Chapter 3.4 --- Summary --- p.77Chapter Chapter 4 --- : Screening the mutant possess higher capacity of forming volatile sulfur compounds (VSCs) from non-starter microbes of sufu product --- p.80Chapter 4.1 --- Introduction --- p.80Chapter 4.2 --- Materials and methods --- p.82Chapter 4.2.1 --- Strains and culture conditions --- p.82Chapter 4.2.2 --- Head space-solid phase microextraction (HS-SPME) analysis --- p.83Chapter 4.2.3 --- Gas Chromatography-Mass Spectrometry (GC-MS) analysis --- p.84Chapter 4.2.4 --- UV mutation --- p.85Chapter 4.2.5 --- Ellman’s method --- p.86Chapter 4.2.6 --- Preparation of cell-free extracts (CFE) for enzymatic assays --- p.86Chapter 4.2.7 --- Enzymatic assay --- p.86Chapter 4.2.7.1 --- L-methionine aminotransferase activity assay --- p.86Chapter 4.2.7.2 --- L-methionine demethiolase activity assay --- p.87Chapter 4.2.7.3 --- α-keto acid decarboxylase activity assay --- p.87Chapter 4.2.7.4 --- C-S lyase activity --- p.88Chapter 4.2.8 --- Statistical analysis --- p.88Chapter 4.3 --- Results and discussion --- p.89Chapter 4.3.1 --- Optimization of SPME extraction condition --- p.89Chapter 4.3.2 --- Selecting the start strain --- p.90Chapter 4.3.4.1 --- Comparison of VSCs production ability --- p.90Chapter 4.3.4.2 --- Comparison of enzymatic activity in L-methionine metabolism --- p.92Chapter 4.3.3 --- Optimization of UV exposure time --- p.95Chapter 4.3.4 --- Screening the mutants --- p.96Chapter 4.3.4.1 --- Comparison of VSCs production ability among the mutants --- p.96Chapter 4.3.4.2 --- Comparison of the L-methionine related enzymatic activities among the mutants --- p.99Chapter 4.3.4.3 --- Identified of strian B-1 --- p.101Chapter 4.4 --- Summary --- p.102Chapter Chapter 5 --- : General conclusions and future work --- p.103References --- p.10