臺灣博碩士論文加值系統

本研究評估一種在台灣被分離出之特有真菌，Tremella flava Chen 之發酵豆奶飲料安全性，雖其機能性效果已在先前研究被證實，然目前並無此產品之動物毒性研究報告，此外該產品是否能降低病原菌感染之研究仍然有限。本研究目標為評估黃白木耳發酵豆奶(TFS)之毒性以及其對BALB/c 小鼠感染臨床分離出之Salmonella typhimurium NJ08.124 之影響。利用Ames test 進行TFS 之微生物基因突變安全性測試時，致突變結果顯示，不論是否S9-mix 添加，不同TFS 濃度(0.25~20.0 mg/plate)之添加均不影響S. typhimurium TA97、TA98、 TA100、 TA102 及 TA1535菌株之生長；以相同方式測試TFS 之抗致突變性，結果顯示其能提高對致突變劑4NQO、mitomycin C 及sodium azide 之抗致突變性並呈現劑量關係。亞急性毒性試驗之進行以黃白木耳發酵豆奶連續餵食BALB/c 小鼠28 天，並觀察低劑量組(0.5 g/kg BW/day)至高劑量組 (2 g/kg BW/day) 對實驗動物之影響。結果顯示餵食TFS後對於其活動力、體重及飲食並無明顯的改變。血清生化質方面，雄鼠及雌鼠之血清檢測項目都在正常範圍內並無顯著差異，而所有組別臟器病理切片也都正常無異。此結果顯示TFS 之無明顯有害效應劑量(NOAEL) 為小於 2 g/kg BW/day。由上述結果顯示，TFS 為一安全性食品且具有保健之潛力。在抗病原菌S.
typhimurium NJ08.124 試驗中，結果顯示小鼠經過TFS 餵食後其免疫球蛋白量 (IgG及IgA)較控制組高，小鼠體內受該病原菌感染情形較對照組為低。本研究證實黃白木耳發酵豆奶可加強免疫力並有效降低S. typhimurium NJ08.124 之感染。

Tremella flava Chen, a novel fungus isolated in Taiwan, was used to ferment soymilk used as beverage. Its beneficial effects have been previously reported in several studies. However, no toxicity reports of this product have been confirmed in animal studies; challenge studies with pathogenic microorganism are still limited. The objective
of this present study was to evaluate potential adverse effects, if any, of Tremella flava fermented soymilk (TFS) and the influence of oral treatment of mice with the fermented soy product on BALB/c mice infected with clinically isolated Salmonella typhimurium NJ08.124. The Ames test employing histidine mutants of the S. typhimurium tester stains TA97, TA98, TA100, TA102 and TA1535 were used to examine the mutagenicity of TFS. No mutagenic activity was observed for either test strains at all used doses (0.25~20.0 mg/plate), with or without S9 activation. The same test was used to examine the ability of TFS to prevent 4-nitroquinoline-1-oxide (4NQO),mitomycin C and sodium azide-induced mutations. The results showed that TFS inhibited mutagenesis caused by the above mutagens was in a dose-dependent manner. TFS was administered to mice daily at levels up to 2 g/kg BW/day for 28 days. No death of the mice caused by the TFS was observed. TFS did not affect the clinical condition of the animals, clinical pathology evaluations, organ weights, and gross necropsy findings. According to the results of the 28-day experiment, the no-observed-adverse-effect-level (NOAEL) for systemic toxicity was suggested to be less than 2 g/kg BW/day for TFS under the conditions of this study. Our findings provide scientific evidence for the safe use and health benefits of TFS. In the infection animal model, fecal excretion and colonization of Salmonella in organs were significantly lowered in mice fed with TFS. Serum IgA and IgG significantly increased in the T and T-S groups compared to control animals. The results of this study suggest that TFS treatment increased the immunity in BALB/c mice challenged with S. typhimurium NJ08.124.

Index to Tables.........................................iv
Index to Figures.........................................v
摘要.....................................................1
Abstract.................................................2
Introduction.............................................3
Literature Review
1. Tremella flava
1.1 Introduction.........................................4
1.2 Functional properties of Tremella....................4
2. Soybean
2.1 Soybean composition..................................6
2.2 Soybean vs. Isoflavones..............................6
3. Isoflavones
3.1 Introduction.........................................7
3.2 Functional properties of isoflavones.................8
4. Safety assessment
4.1 Categories of health foods...........................9
4.2 Toxicity tests......................................10
5. Immunity
5.1 Introduction........................................14
5.2 Cytokines...........................................15
5.3 Lipopolysaccharide..................................15
5.4 Concanavalin A......................................15
Objective...............................................17
Experiment design.......................................18
Materials and methods
1. Materials............................................19
1.1 Soybean.............................................19
1.2 Microbial strains...................................19
1.3 Animal..............................................19
1.4 Nutrient Agar.......................................19
1.5 Nutrient agar plates................................20
1.6 Cell culture media..................................23
1.7 Chemicals...........................................23
1.8 Apparatus...........................................25
2. Methods
2.1 Production of Tremella flava fermented soymilk......26
2.2 Chemical composition of Tremella flava fermented soymilk (TFS) powder....................................26
2.3 Toxicity study
2.3.1 Ames test.........................................28
2.3.2 Subacute (28-day) gavage toxicity study...........30
2.4 Influence of Salmonella typhimurium NJ08.124 infection in BALB/c mice
model using Tremella flava fermented soymilk............36
2.4.1 Fermented soymilk (TFS) powder....................36
2.4.2 Bacterial strain (Pathogen).......................36
2.4.3 Animals...........................................36
2.4.4 Administration....................................36
2.4.5 Experimental conduct..............................36
2.4.6 Specific immunity test............................37
2.4.7 Preparation of splenocytes........................37
2.4.8 Anti-infection test...............................38
2.4.9 Cytokine immune assay.............................38
2.4.10 Non-specific antibody immune assay...............38
3. Statistical analysis.................................39
Results and Discussion
1. Composition analysis.................................40
2. Toxicity study
2.1 Confirmation of Salmonella typhimurium strain genotype (Ames test).............................................40
2.2 Toxicity test.......................................44
2.3 Mutagenicity test...................................44
2.4 Anti-mutagenicity test..............................45
2.5 Subacute toxicity (28-day) gavage study
2.5.1 Survival, clinical observations and body weights..50
2.5.2 Feed and water consumption........................50
2.5.3 Serum chemistry...................................54
2.5.4 Visceral organ weights............................54
2.5.5 Macroscopic and microscopic examinations..........58
2.5.6 Cytokine secretions of splenocytes................58
2.5.7 Human equivalent dose (HED) calculations..........59
3. Animal infection model with Salmonella typhimurium NJ08.124
3.1 Survival, body weights and feed consumption.........68
3.2 TFS vs. Non-specific immunity in BALB/c mice........68
3.3 Anti-infection test.................................77
Conclusion..............................................80
Future Prospects........................................82
References..............................................83

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