臺灣博碩士論文加值系統

豆豉為我國傳統之大豆發酵產品，可藉由微生物分泌酵素對大豆進行水解， 使其產生獨特之風味，常作為食品調味之用，也是許多發酵豆製品之起源。豆腐 乳為廣受國人歡迎之豆類發酵食品，具有特殊之風味及質地。鹹蛋白由於其鹽分 含量較高利用性較低，行政院農業委員會畜產試驗所開發以鹹鴨蛋白為基質，接 種經活化之豆豉麴，經發酵熟成後製成之「鹹蛋白腐乳」(salted egg white sufu) 產 品接受性高，惟其製程中微生物菌相變化尚不清楚，故本研究室與畜試所合作， 對此產品進行菌相之研究。試驗一旨在運用總菌數培養基 (plate count agar, PCA) 與真菌通用之馬鈴薯葡萄糖瓊脂培養基 (potato dextrose agar, PDA) 分離及保存豆 豉中微生物，並以 16S rDNA、部分 18S rDNA、黃麴毒素合成基因 C1 區及 omtA 基因來鑑定豆豉 (鹹蛋白腐乳之菌元) 中微生物之種類；試驗二則另以基因選殖 (gene cloning) 方法鑑別製程中之豆豉、腐乳半成品 (發酵 5 天) 及腐乳完成品 (發 酵 5 天及熟成 14 天) 之菌相變化。 結果顯示：試驗一中共可發現 9 種不同外觀型態之菌落，經分離各菌落及單 獨培養並鑑定其品種，結果顯示細菌類主要為芽孢桿菌屬 (Bacillus)，其中包含 Bacillus amyloliquefaciens 、 Bacillus licheniformis 、 Bacillus methylotrophicus 及 Bacillus subtilis 等，另有部分之腸球菌屬 (Enterococcus) 菌株；真菌以麴黴屬 (Aspergillus) 中之 Aspergillus oryzae 與 Aspergillus terreus 為主，此結果與許多發酵 豆類食品相似。試驗二結果顯示豆豉中細菌亦是以 Bacillus 為主，佔全體總數比例 之 88.9%，其中包含少許試驗一中未被檢出之 Bacillus sp.、Enterococcus durans 及 Staphylococcus. sp.等；真菌則以 A. oryzae 為主，所含之菌株與試驗一相似。腐乳 半成品中之 Bacillus 屬佔全體總數比例減少至 51.0%，其中豆豉中主要菌株 B. amyloliquefaciens 與 B. subtilis 大幅減少至 6.1%與 14.3%；反之，腸球菌屬及葡萄 球菌屬 (Staphylococcus) 比例分別大幅增加至 16.3%及 20.4%，另外亦於半成品中 檢測出 Enterococcus faecium、Enterococcus lactis 、 Staphylococcus carnosus 及 Terribacillus 等細菌菌株及 uncultured fungus clone 真菌菌株。腐乳成品中 Bacillus 菌株重回優勢，推測可能是因為其具有產生內孢子 (endospore) 之能力，可以存活 於含有較高鹽分及酒精之環境有關，故 E. lactis 及 uncultured Staphylococcus sp. clone 未被檢出，成品中真菌部分僅有檢出 A. oryzae，雖然該菌株非豆豉中主要優 勢菌，然而於最終產品中大量增加，推測該菌較能適應鹹蛋白之基質。綜觀上述， 運用分子技術確實可以快速準確地鑑定鹹蛋白腐乳中微生物種類及菌相，儘管發 酵階段時菌相有較大之變動，但仍是以 Bacillus 與 A. oryzae 為主要之優勢菌。傳 統人工培養法具有可以保留純種菌株之優點，但是亦存在著較費工費時與菌株分 離可能遺漏等問題，因此可依據試驗目的選擇適當操作方法來進行。

Douchi, a traditional fermented soybean product in Taiwan, China, and other areas, as hydrolyzed by the microbial enzymes and producing some unique flavors makes it to be applied as a food seasoning agent and also be the raw materials of manufacturing many other fermented soybean products for years. Sufu, which is a fermented soybean product, is also very popular in many Asian areas due to its subtle textures and unique flavors. Due to a higher salt content, salted egg white is under-utilized. Therefore, the Livestock Research Institute (LRI), Council of Agriculture, Executive Yuan, has been developing a ‘‘salted egg white sufu” which was manufactured using salted egg white as a substrate, inoculated with douchi koji, fermented, and ripened. This product is well-accepted, yet its microbial changes during the processing have not been elucidated. Therefore, the objective of the experiment 1 was to isolate, preserve, and molecular identify the microbial strains in douchi by using the plate count agar (PCA), potato dextrose agar (PDA), and 16S, 18S ribosomal RNA gene (rDNA), aflatoxin gene of C1 region and omtA gene methods. The objective of the experiment 2 was to detect the relative abundance of the microorganisms in douchi, semi-finished (fermented for 5 days) and final products (fermented for 5 days and ripened for 14 days) of the salted egg white sufu by the gene cloning method. In the exp. 1, by isolating and identifying the different pure cultures, the results showed that in douchi there were 9 types of colonies with different morphological characteristics which included bacterium of Bacillus amyloliquefaciens, Bacillus licheniformis, Bacillus methylotrophicus, Bacillus subtilis, Enterococcus sp. etc. and fungus of Aspergillus oryzae and Aspergillus terreus. This result was consistent with those observed from the other soybean-fermented foods. In the exp. 2, by the cloning method, it was found Bacillus also dominated in douchi (88.9%) and some strains of Bacillus sp., E. durans and Staphylococcus sp. which were not observed in exp. 1 were detected. Similar to that found in exp. 1, A. oryzae was the dominant fungus detected in douchi. In the semi-finished product, the percentage of Bacillus was reduced to 51.0% and the percentages of B. amyloliquefaciens and B. subtilis were reduced remarkably to 6.1% and 14.3%, respectively. Contrarily, the percentages of Enterococcus and Staphylococcus were increased to 16.3% and 20.4% remarkably. Besides, E. faecium, E. lactis, S. carnosus and Terribacillus and uncultured fungus clone 1016RhFu were the new strains that detected in the semi-finished product. In the final product, Bacillus returned to dominate probably because it can produce some endospores which can survive in a harsh environment with comparatively high salt and ethanol contents. This might also be the reason why E. lactis and uncultured Staphylococcus sp. clone were
not detected. A. oryzae was the only fungi detected in the final product, in which A. oryzae RIB40 became dominant in salted egg white probably due to its adaption of the environment. In conclusion, our result demonstrated that the molecular techniques enable to identify microorganisms and recognize the relative abundance of the microbial strains in the salted egg white sufu faster and more accurately. The relative abundance of the microorganisms might be changed during the processing; however, Bacillus and A. oryzae still dominated. Through the artificial culturing, it is possible to obtain the pure strains from samples; however, some microorganisms might be possibly omitted during the selection of microorganisms. Properly selection the methods base on the needs of experiment should be addressed.

壹、前言 ...........1
貳、文獻檢討 ...........2
一、豆豉介紹...........2
(一) 豆豉的歷史文化...........2
(二) 豆豉的營養價值...........2
(三) 豆豉的分類 ...........2
(四) 豆豉的生產工藝...........4
(五) 豆豉製作過程微生物的變化...........5
(六) 豆豉生產中存在之瓶頸...........5
二、蛋白質發酵食品之微生物種類...........6
(一) 發酵豆類食品之微生物種類...........6
(二) 發酵肉品之微生物種類...........6
三、微生物之蛋白酶產生與應用...........8
(一) 麴菌屬蛋白酶之產生及應用...........8
(二) 嗜鹽細菌蛋白酶之產生及應用...........9
四、米麴菌之鑑定...........10
五、分子生物技術之應用...........11
(一) 聚合酶鏈鎖反應...........11
(二) 核糖體 DNA 之應用...........13
(三) 基因選殖 (gene cloning) ...........14
(四) 多源基因體 (metagenomics) ...........14
參、材料與方法 ...........16
試驗一、豆豉菌株分離與分子技術鑑定...........16
(一) 豆豉來源...........16
(二) 培養基與培養液配製 ...........16
(三) 菌株之分離純化...........16
(四) 顯微鏡觀察 ...........17
(五) 分離菌株之分子技術鑑定...........18
1.菌株 genomic DNA 之萃取...........19
2.PCR 擴增 16S 與 18S rDNA 片段 ...........20
3.PCR 產物純化...........21
4.DNA 序列定序與資料庫比對...........21
5.米麴菌之鑑定...........23
試驗二：鹹蛋白腐乳製作過程之菌相變化...........26
(一) 豆豉及鴨蛋來源...........26
(二) 鹹蛋白腐乳之製作...........26
1.鹹鴨蛋醃製...........26
2.鹹蛋白塊之製備...........26
3.種麴製作...........27
4.鹹蛋白腐乳發酵及熟成 ...........27
5.鹹蛋白腐乳取樣與前處理方法...........28
(三) 菌相變化之分子技術鑑定...........30
1. 菌株 genomic DNA 之萃取...........31
2. PCR 擴增 16S 與 18S rDNA 片段...........32
3. PCR 產物純化...........34
4. 基因選殖 (gene cloning) ...........34
5. DNA 序列定序與資料庫比對 ...........37
肆、結果與討論...........38
試驗一、豆豉菌株之分離與鑑定 ...........38
(一) 菌株分離純化...........38
(二) 分離菌株外觀型態之肉眼與顯微觀察 ...........38
(三) 分離菌株之分子鑑定...........42
1. PCR 擴增 16S 與 18S rDNA 片段...........42
2. DNA 序列定序、米麴菌之鑑定與資料庫比對 ...........43
試驗二、鹹蛋白腐乳製作過程之菌相變化...........48
(一) 菌相變化之分子鑑定...........48
菌株 genomic DNA 之萃取及 PCR 擴增 16S 與 18S rDNA 片
段...........48
2. 基因選殖 (gene cloning) ...........49
3. DNA 序列定序與資料庫比對...........52
伍、結論...........74
陸、參考文獻： ...........75
柒、附錄...........82

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