臺灣博碩士論文加值系統

乳酸菌 (Lactic acid bacteria, LAB)被廣泛地使用在製造各類乳製品，是動植物正常菌群之一，亦是probiotics之一，且被歸類為安全食用菌(GRAS)。傳統植物性醱酵製品分離之乳酸菌與乳酪、醱酵乳及肉品醱酵製品中的乳酸菌有所差異，而乳酸菌在乳製品及醱酵工業的經濟效益甚大，故篩選具probiotic特性或具特殊功能的乳酸菌有其必要性。本研究探討不同種類的台灣本土性醱酵蔬菜之含菌量及菌種鑑定。經隨機採集7件市售醱酵蔬菜(泡菜5件、酸筍1件及酸菜1件)，以含有CaCO3之GYP培養基進行乳酸菌檢測，結果泡菜中含乳酸菌量大於酸菜及酸筍食品，其所含乳酸菌量平均達107 CFU/mL以上。從分離相挑選288個產酸能力強的菌落進行菌種鑑定，結果鑑定出Lactobacillus fermentum、Lactobacillus casei、Lactobacillus acidophilus、Lactococcus plantarum、Lactococcus lactis等乳酸菌菌種。此303個菌落針對耐酸性(pH2-pH3)及耐膽鹽性(0.2%、0.3%及0.4% bile slats)，篩選出33株耐受性較佳的乳酸分離菌株。針對此33株菌株進行模擬消化過程分析、腸道吸附能力(adhesion in Caco-2 cell)、抑制腸道細菌、醱酵纖維寡糖能力等特性篩選出6株能忍受消化過程傷害、吸附至腸道細胞、抑制腸道害菌生長及代謝異麥芽寡糖、龍膽寡糖等probiotics特性的乳酸菌分離株。其分別為Lactococcus plantarum strain E38、E46、E51及Lactobacillus casei strain E7、E15、V38。

Most lactic acid bacteria (LAB) are generally recognized as safe (GRAS) for human consumption, and used world wide in fermented dairy. LAB are widely distribution among animals and plants. Some of them are also considered as probiotics. LAB isolated form traditional fermented vegetables are different from that of cheese, yogurt, and fermented meat. Because LAB have large economic benefits in the industrial of fermented food products and milk products, it is important to screen LAB which have probiotics properties or specific functions. The purpose of this study was to survey the lactic acid bacteria flora of some Taiwanase local fermented vegetables and tried to find some LAB as usable probiotics. Total LAB counts of seven randomly sampled fermented vegetables were analyzed. The average of total LAB counts in five brands of chinese pickled vegetables were over 107 CFU/mL, however one brand of acidified vegetables and acidified bamboo shoot were less than that of pickled vegetables. Among the isolated LAB flora, 288 colonies with high acid producing ability were selected for further analysis. Through biochemical and microbial tests, 147 of 288 colonies were identified as Lactobacillus casei, 6 of 288 colonies were identified as Lactobacillus fermentum, 37 of 288 colonies were identified as Lactobacillus acidophilus, 94 of 288 colonies were identified as Lactococcus plantarum, and 4 of 288 colonies were identified as Lactococcus lactis. Through acid tolerance (pH 2.0, pH 3.0) and bile salts tolerance (0.2%、0.3%、0.4% bile salts) assays, 33 of 288 colonies were selected as high acid and bile salt tolerants. Analysis of the 33 colonies with simulated digestive tract analysis, adhere to Caco-2 cell, antimicrobial activity assay, and oligosaccharides digestiable assay, 6 colonies of Lactococcus plantarum strain E38, E46, E51 and Lactobacillus casei strain E7, E15,V38 were found to be could tolerant from digestive system, could adhere to Caco-2 cell, could restrain pathogen bacteria growth,and could digest isomaltooligosaccharides and gentiooligosaccharides.

第一章 前言……………………………………………………………..1
1.1 緒言………………………………………………………………….1
1.2植物和植物材料中的乳酸菌………………………………………...2
1.3 益生菌之定義及特性……………………………………………….6
1.4乳酸菌的定義………………………………………………………...7
1.5 probiotics乳酸菌之篩選研究…………………………………….…8
1.6 Probiotics乳酸菌之醫藥功效……………………………………...11
1.7 Prebiotics定義及特性……………………………………………...15
1.8 Synbiotics的定義…………………………………………………..17
1.9本研究的動機與目的……………………………………………….17
第二章 材料與方法……………………………………………………20
2.1 材料………………………………………………………………...20
2.1.1 醱酵蔬菜來源………………………………………………...….20
2.1.2 化學試劑與酵素………………………………………………....20
2.1.3 套組………………………………………………………………20
2.1.4儀器………………………………………………………………..21
2.2 方法………………………………………………………………...21
2.2.1 醱酵蔬菜食品所含乳酸菌之篩選及鑑定……………………21
2.2.1.1使用菌株與培養基………………………………………...21
2.2.1.2醱酵蔬菜食品所含乳酸菌之篩選……………………...…21
2.2.1.3菌種鑑定方法……………………………………………...22
2.2.1.4 API菌種鑑定……………………………………………...24
2.2.2 Probiotics特性分析…………………………………………...25
2.2.2.1乳酸菌分離株耐酸性分析………………………………..25
2.2.2.2乳酸菌分離株耐膽鹽性分析……………………………..25
2.2.2.3模擬消化程序之處理…………………………………...…26
2.2.2.4 Caco-2細胞株培養………………………………………..27
2.2.2.5乳酸菌分離株吸附性分析………………………………...27
2.2.2.6乳酸菌分離株抑制腸道害菌分析………………………...28
2.2.2.7乳酸菌分離株抑制腸道益菌分析………………………...29
2.2.3 Prebiotics特性分析……………………………………………29
2.2.3.1 Prebiotics-MRS plate之製備…………………………….29
2.2.3.2 Prebiotics-MRS plate之分析…………………………….30
2.2.3.3乳酸菌分離株在含prebiotics物質培養基之生長曲線分析…………………………………………………………..30
2.2.3.4 乳酸菌分離株代謝prebiotics物質之成分分析………...31
第三章 結果……………………………………………………………32
3.1 醱酵蔬菜所含乳酸菌量及菌種之分析…………………………...32
3.1.1乳酸菌含量檢測………………………………………………..32
3.1.2乳酸菌菌種鑑定……………………………………………..…32
3.2乳酸菌分離株probiotics特性分析………………………………...33
3.2.1耐膽鹽性評估…………………………………………………..33
3.2.2耐酸性評估……………………………………………………..33
3.2.3模擬消化過程之評估…………………………………………..34
3.2.4吸附性評估……………………………………………………..35
3.2.5吸附因子分析…………………………………………………..35
3.2.6抑菌性評估……………………………………………………..35
3.3乳酸菌分離株醱酵寡糖特性分析………………………………….37
3.3.1含寡糖之MRS plate分析……………………………………..37
3.3.2乳酸菌分離株在含寡糖之生長曲線分析……………………..37
3.3.3乳酸菌分離株代謝寡糖之成分分析…………………………..38
3.4 特異性probiotics乳酸菌之篩選……………………………….....38
第四章 討論……………………………………………………………40
4.1醱酵蔬菜所含乳酸菌量及菌種之探討…………………….………40
4.2乳酸菌對消化過程之探討………………………………………….41
4.3乳酸菌吸附能力之探討…………………………………………….43
4.4乳酸菌抑菌性之探討……………………………………………….44
4.5乳酸菌醱酵寡糖能力之探討……………………………….………45
第五章 結論…………………………………………………………....47
參考文獻………………………………………………………………..48
表目錄
表一、人類胃腸道分佈之菌群…………………………………………58
表二、特異性益生菌篩選策略…………………………………………59
表三、醱酵蔬菜來源與乳酸菌含量……………………………………60
表四、API菌種鑑定結果……………………………………………….61
表五、乳酸菌分離株之膽鹽耐受性分析………………………………62
表六、乳酸菌分離株之耐酸性分析……………………………………65
表七、乳酸菌分離株之耐酸性與耐膽鹽性分析………………………67
表八、乳酸菌分離株之吸附性分析……………………………………68
表九、不同處理對於乳酸菌吸附之影響………………………………70
表十、乳酸菌分離株之菌液抑制腸道害菌分析………………………71
表十一、中和後之乳酸菌分離株菌液抑制腸道害菌分析……………73
表十二、乳酸菌分離株菌液抑制腸道益菌分析………………………75
表十三、乳酸菌分離株之醱酵一些寡糖之析………………………....77
圖目錄
圖一、果寡糖之化學結構及反應機制…………………………………79
圖二、異麥芽寡糖之化學結構及反應機制……………………………80
圖三、API system製作流程……………………………………………81
圖四、Caco-2細胞株生長型態…………………………………………82
圖五、不添加乳酸菌之Caco-2細胞株………………………………...83
圖六、Lactobacillus GG附著於Caco-2 細胞株………………………84
圖七、Lactococcus plantarum E51附著於Caco-2細胞株…………...85
圖八、以含有寡糖之MRS plate進行醱酵寡糖特性分析模式圖…….86
圖九、乳酸菌分離株在含寡糖培養基之生長曲線分析………………87
圖十、果寡糖標準品之HPLC管住層析分析結果……………………88
圖十一、異麥芽寡糖標準品之HPLC管住層析分析結果……………89
圖十二、龍膽寡糖標準品之HPLC管住層析分析結果………………90
圖十三、以HPLC分析乳酸菌分離株利用果寡糖結果………………91
圖十四、以HPLC分析乳酸菌分離株利用異麥芽寡糖結果…………92
圖十五、以HPLC分析乳酸菌分離株代謝龍膽寡糖結果……………93

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