臺灣博碩士論文加值系統

豆漿與豆腐是高水活性、低酸性且高蛋白質含量的黃豆食品，微生物容易在其中快速生長而造成腐敗或食品中毒，故殺菌條件與儲藏環境將影響產品之品質及其保存期限。本研究目的為分離並鑑定豆漿與豆腐中常見的微生物，作為製程中微生物控制、保存期限評估及品管檢驗的參考。以市售經超高溫瞬間殺菌之豆漿與商業殺菌之豆腐為樣品進行5 ℃、15 ℃、25 ℃與35 ℃儲藏試驗，結果顯示豆漿中微生物為隨機出現，而出現頻率最高者為Rhodococcus，而豆腐的微生物在儲藏過程有明顯生長，其中以Bacillus與Paenibacillus最廣泛存在且為腐敗時的優勢菌。針對上述分離之微生物進行特性分析，發現Rhodococcus、Bacillus與Paenibacillus皆可在中性食品中生長良好，高於冷藏溫度時Rhodococcus會在豆漿中生長，但其沒有致病性基因與抗生素抗性，且在低溫儲藏下會逐漸死亡。另一方面，高於冷藏溫度時Bacillus會在豆腐中快速生長，其可能在腸道中產生腸毒素且對抗生素具有抗性。然而Paenibacillus在低溫下可生長，雖然不具有致病性與抗生素抗性，仍會造成豆腐的腐敗。此外，Rhodococcus不耐巴氏德殺菌，因此推測其為殺菌後的汙染，但Bacillus與Paenibacillus在加熱後皆可存活，故其可能為殺菌前就存在原料中或在加工設備中殘留而反覆汙染。將Rhodococcus接種回豆漿與培養基中建立其生長預測模式，發現在15 ℃至35 ℃之間其生長速度隨溫度上升而愈來愈快，且豆漿為適合其生長的基質。另一方面，以16S rDNA微生物體分析豆腐在15 ℃腐敗過程的菌相變化，確認其優勢菌為Bacillus與Paenibacillus，以real-time qPCR偵測豆腐中的Paenibacillus，雖專一性與敏感度尚有改善空間，仍是一有潛力的快速檢驗方法。應用real-time qPCR配合傳統培養法檢測冷藏下腐敗的其他市售豆腐，發現Paenibacillus可能是存在於許多冷藏豆腐中，其來源與檢驗值得注意與探討。

Microorganisms in soymilk and tofu can grow fast and cause spoilage or foodborne disease because of their high water activity, low acidity and high protein content. Therefore, the condition of sterilization and storage can affect the quality and shelf-life of products. The aim of this study was to isolate, identify and characterize the common microorganisms in commercial soymilk and tofu to provide reference for microbial control during process, shelf-life evaluation and quality control test. Commercial ultra-high temperature (UHT) soymilk and tofu with commercial sterilization were conducted storage assay including 5 ℃, 15 ℃, 25 ℃ and 35℃, and the results showed occurrence of microorganisms in soymilk were random and Rhodococcus were the most often found microorganism in storage assay. On the other hand, the bacterial concentration in tofu increased during storage period, furthermore, Bacillus and Paenibacillus were the dominant genera while spoilage and widely present in tofu. The isolates from soymilk and tofu were characterized and found Rhodococcus, Bacillus and Paenibacillus could grow well in neutral environment. Although Rhodococcus could grow at room temperature, they gradually died under refrigeration and without virulence gene or antibiotic resistance. Bacillus could grow fast when the temperature higher than refrigeration and may be able to produce enterotoxin in gastrointestinal tract and have resistance to antibiotic. However, Paenibacillus could grow under low temperature and result in tofu spoilage although they didn’t have toxin gene and antibiotic resistance. In addition, Rhodococcus couldn’t survive after pasteurization consequently assuming they contaminated after UHT process. Bacillus and Paenibacillus could survive after pasteurization and had potential to form biofilm. Hence, they may exist in materials and survived after commercial sterilization or on the food-contact surface during process and cause wide contamination. Rhodococcus were inoculated to soymilk to develop predictive growth model. The results showed Rhodococcus could grow faster as temperature rose between 15 ℃ and 35 ℃ and soymilk is a suitable substrate for Rhodococcus. The microbiota succession of tofu during storage at 15 ℃ were analyzed to reconfirm Bacillus and Paenibacillus were the dominant genera in tofu. Because of the importance of Paenibacillus in tofu, a potential rapid detection method using real-time qPCR was developed though the specificity and sensitivity were still need to be improved. Tofu from other manufacturers were examined by real-time qPCR and traditional method and found Paenibacillus may present in many refrigerated tofu.

謝誌 i
摘要 ii
Abstract iii
目錄 v
圖目錄 viii
表目錄 ix
第一章 前言 1
第二章 文獻回顧 2
第一節、 豆漿與豆腐 2
一、 黃豆營養成分及其製品 2
二、 豆製品之微生物規範 3
三、 曾發現之微生物 3
第二節、 微生物檢驗與鑑定 6
一、 傳統方法 6
二、 16S rDNA鑑定 6
三、 基質輔助雷射脫附游離飛行時間式質譜儀 7
四、 即時聚合酶連鎖反應 7
五、 微生物體分析 8
第三節、 食品保存期限與腐敗 10
一、 食物浪費 10
二、 食品腐敗 10
三、 食物保存期限評估 10
第四節、 預測微生物學 12
一、 簡介 12
二、 一級模式 12
三、 二級模式 13
四、 三級模式 13
五、 預測微生物學之應用 14
第五節、 Rhodococcus、Bacillus與Paenibacillus 16
一、 基本特性 16
二、 與食品之關係 17
第三章 研究目的與架構 19
第四章 材料與方法 21
第一節、 材料 21
一、 樣品 21
二、 實驗菌株 21
三、 藥品 23
四、 微生物培養基與稀釋液 24
五、 儀器與設備 24
六、 套裝軟體 25
第二節、 實驗方法 26
一、 腐敗性微生物分離與鑑定 26
二、 菌株特性分析 30
三、 微生物生長預測模式 35
四、 以非培養法檢測豆腐之Paenibacillus 37
第五章 結果 40
第一節、 腐敗性微生物分離與鑑定 40
一、 豆漿儲藏試驗 40
二、 豆腐儲藏試驗 40
三、 分離菌株鑑定 41
第二節、 菌株特性分析 48
一、 豆漿分離之Rhodococcus spp.與Dietzia sp. 48
二、 由豆腐分離之Bacillus spp.與Paenibacillus spp. 54
三、 推測微生物來源及對黃豆產品的影響 59
第三節、 微生物生長預測模式 64
一、 一級模式與二級模式建立 64
二、 模式驗證與評估 65
第四節、 以非培養法檢測豆腐之Paenibacillus 72
一、 16S rDNA微生物體分析 72
二、 以real-time qPCR檢測豆腐中的Paenibacillus 74
三、 其他市售豆腐檢測 80
第六章 討論 83
第一節、 腐敗性微生物分離與鑑定 83
一、 豆漿儲藏試驗 83
二、 豆腐儲藏試驗 83
三、 分離菌株鑑定 84
第二節、 菌株特性分析 86
一、 豆漿分離之Rhodococcus spp.與Dietzia sp. 86
二、 由豆腐分離之Bacillus spp.與Paenibacillus spp. 87
三、 推測微生物來源及對黃豆產品的影響 89
第三節、 微生物生長預測模式 93
一、 一級模式與二級模式建立 93
二、 模式驗證與評估 94
第四節、 以非培養法檢測豆腐之Paenibacillus 96
一、 16S rDNA微生物體分析 96
二、 以real-time qPCR檢測豆腐中的Paenibacillus 96
三、 其他市售豆腐檢測 98
第七章 結論與展望 100
第八章 參考文獻 101
附錄 120
APPENDIX 132

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