臺灣博碩士論文加值系統

已知去醣基大豆異黃酮及胜肽具抗肥胖活性，並可促進細胞吸收葡萄糖。本研究以 Lactobacillus sp. FPS 2520 (FPS 2520) 及 Bacillus sp. N1 (N1) 為測試菌株，首先探討各別菌株發酵豆粕懸浮液能力，及所得去醣基大豆異黃酮 Daidzein 及 Genistein，在基質與水含量比 1:8 之豆粕懸浮液中 FPS 2520 靜置培養 24 小時再繼續振盪培養 48 小時，所得發酵液之游離胺基態氮 (FAN)、總胜肽 (TP)、Daidzein 及 Genistein 含量分別為 5.36 ± 0.09 與 4.77 ± 0.58 mg / mL 及 4.43 ± 0.14 與 3.06 ± 0.08 mM。N1 菌株經連續振盪 72 小時所得發酵液之 FAN、TP、Daidzein 及 Genistein 含量分別為 9.77 ± 0.27 與 8.80 ± 1.97 mg / mL 及 4.33 ± 0.11 與 3.21 ± 0.11 mM。混株培養模式先接種 N1 振盪培養 24 小時，再接種 FPS 2520 繼續培養所得 FAN、TP、Daidzein 及 Genistein 含量分別為 7.31 ± 0.89 與 5.97 ± 0.57 mg / mL 及 3.93 ± 0.60 與 2.97 ± 0.31 mM。但若於接種 FPS 2520 24 小時後將溫度提高再發酵之二階段發酵模式可顯著提高活性物質含量，發酵 24 小時所得 FAN、TP、Daidzein 及 Genistein 含量分別為 7.75 ± 0.09 與 6.38 ± 0.14 mg / L 及 4.50 ± 0.29 與 3.37 ± 0.01 mM 與未發酵豆粕相比混和菌株二階段發酵產物所含抗營養因子植酸及皂素含量為未發酵豆粕之 69.2% 及 35.9%，且發酵產物支鏈胺基酸 Leucine 含量增加 33.0%。二階段溫度之複合菌株發酵產物能顯著降低 3T3 – L1 脂肪細胞 TC 與 TG 其含量分別為控制組的 59.8% 及 35.9%，其乃藉由抑制細胞 Acetyl-CoA carboxylase (ACC) 蛋白質表現量，並且亦能啟動 3T3 – L1 脂肪細胞中 Protein kinase B (AKT) 訊息蛋白進而使 Glucose transporter type 4 (GLUT 4) 表現量增加。在有胰島素及無胰島素下發酵產物均能顯著提升 FL83B 小鼠肝臟葡萄糖利用率，分別為控制組的 179.5% 及 128.6%。綜合上述，豆粕經由 Lactobacillus sp. FPS 2520 和 Bacillus sp. N1 發酵能提升去醣基異黃酮、胜肽含量及改善胺基酸組成，並降低抗營養因子以製備具抑制脂質累積及促進葡萄糖利用率潛力之飼料添加劑

Isoflavone aglycosides and soy peptides have been demonstrated to have anti-obesity activity. The aim of this study is to ferment soybean meal (FSBM) to increase the contents of isoflavone aglycosides, peptide and amino acid for the preparation of nutritional feed additive with anti-obesity potential. Bacillus sp. N1 and Lactobacillus sp. FPS 2520 were used as the tested strains in this study. During fermentation period, soybean meal suspension was incubated by FPS 2520 and stand 24 hr then shaking at 60rpm. The contents of Daidzein, Genistein, free amino nitrogen (FAN) and total peptide (TP) were 4.43 ± 0.14 mM, 3.06 ± 0.08 mM, 5.36 ± 0.09 and 4.77 ± 0.58 mg / mL, respectively. N1 strain was incubated with shaking for 72 hr, the contents of daidzein, genistein, FAN and TP were 4.33 ± 0.11 mM, 3.21 ± 0.11 mM, 9.77 ± 0.27 mg / mL, 8.80 ± 1.97 mg / mL, respectively. SBM fermented products by N1 and FPS2520 with using two stages of incubation temperatures (FSBM - M) could increase contents of genistein. The contents of Daidzein, Genistein, FAN and TP were 4.50 ± 0.29 mM, 3.37 ± 0.01 mM, 7.75 ± 0.09 and 6.38 ± 0.14 mg / mL,The residual persentages of phytateand saponin in FSBM - M were 69.2% and 20%,respectively,compared to unfermented raw SBM.In addition, the content of the branch – chain amino acid, leucine in FSBM - M was increased by 33.0%. FSBM - M extract could inhibit the Acetyl – CoA carboxylase (ACC) expression that significantly decreased the contents of total cholesterol and triglyceride in differentiated 3T3 – L1 adipocytes by 64.1% and 68.2%, respectively. FSBM - M extract also could enhance Phospho – protein kinase B (p - AKT) and increase glucose transporter type 4 (GLUT 4) in 3T3 – L1. The glucose uptake percentages of FL83B hepatocytes in the presence of FSBM extract with 10 μM insulin or without insulin were increased to 179.5% and 128.6%, respectively, compared to control cells. In summary, fermentation of SBM by using the two – stage fermentation model of co – cultures of FPS 2520 and N1 could increace the contents of Daidzein, Genistein, FAN and total peptide, and decreace the contents of phytate and saponin. The obtained FSBM was demonstrated to be a feed nutritional additive with hypolipidemic and hypoglycemic activity.

摘要I
AbstractII
目錄III
圖目錄V
表目錄VII
附錄VIII
壹、前言 1
貳、文獻整理3
一、飼料添加劑 (Feed nutrient additive)3
1.飼料添加劑及其功能3
2.飼料添加物之分類3
3.飼料添加劑之抗氧化活性5
4.飼料添加劑適口性及改善動物腸胃功能5
5.飼料添加劑之促進動物生長性5
二、黃豆 (Soybean)6
1.黃豆簡介6
2.豆粕6
3.發酵提升豆粕營養價值6
三、乳酸菌與納豆菌介紹7
1.乳酸菌 (Lactic acid bacteria) 定義及特性7
2.乳酸菌之益生功效7
3.納豆菌 (Bacillus subtilis natto) 定義及特性8
4.納豆菌之益生功效8
三、肥胖 (Obesity)9
1.肥胖的成因9
2.脂肪細胞種類與特性9
3.脂肪代謝蛋白與合成蛋白10
4.脂肪組織與肥胖10
5.肥胖與胰島素阻抗11
6.抗肥胖活性物質11
參、實驗設計13
肆、實驗材料與方法14
一、實驗材料14
1.原料14
2.實驗菌株14
3.化學藥品14
二、儀器設備14
三、實驗方法15
1.菌株保存15
2.菌株活化15
3.單株菌發酵條件探討16
4.複合菌株一階段發酵條件探討17
5.複合菌株二階段發酵條件探討17
6.以細胞模式分析發酵液抑制脂質累積及促進葡萄糖利用率活性分析17
7.西方墨點法分析20
8.分析22
伍、結果與討論29
1.乳酸菌 FPS 2520 單株發酵最佳條件探討29
2.Bacillus sp. N1 單株發酵最佳條件探討31
3.複合菌株發酵最佳條件探討33
4.複合菌株二階段發酵最佳條件探討34
5.以高效能液相層析儀 (HPLC) 分析豆粕發酵液中異黃酮含量之探討34
6.經發酵後豆粕懸浮液之一般組成分、營養及抗營養物質與胺基酸組成變化35
7.利用揮發性鹽基態氮 (volatile basic nitrogen，VBN) 分析豆粕發酵液中改善風味之探討35
8.以小鼠脂肪細胞 3T3 – L1 模式分析乳酸菌及納豆菌單株發酵豆粕均質液萃取物抑制脂質累積能力36
9.以西方墨點法 (Western blot) 分析複合菌株發酵豆粕懸浮液萃取物與小鼠脂肪細胞 3T3 – L1 細胞之 p – AKT (p - PKB)、GLUT 4 及 p – ACC 的蛋白質表現量38
10.以小鼠肝臟細胞 FL83B 模式分析乳酸菌及納豆菌單株發酵豆粕懸浮液萃取物促進葡萄糖利用率能力38
陸、結論40
伍、參考文獻41
陸、圖表54

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