臺灣博碩士論文加值系統

摘要

以黃豆為原料製成豆乳，利用混合乳酸菌於42℃下進行發酵20小時後，將發酵豆乳中之乳清部分以凍結乾燥法製成粉末，探討不同發酵方式製成的乳清粉末對血管升壓素轉換酶(ACE)的抑制活性與其生理活性物質的影響。豆乳發酵20小時後，單獨發酵(SF組)與混合發酵之豆乳乳清(SFH組)的pH值分別由6.55降至4.41與4.15，而酸度由0.06%分別增至0.23與0.91%。SFH組的可溶性蛋白質由62.2增加為236.7 mg/g為SF組的六倍。胜肽方面，SFH組的375.2 mg/g為SF組的十倍。游離胺基酸總量以SFH組的16786.74 mg/100g 高於SF組的2088.44 mg/100g，且其中γ-aminobutyric acid (GABA)也以SFH組的201.05 mg/100g高於SF組的136.1 mg/100g，異黃酮中Daidzein與Genistein也以SFH組(246.24與188.15 μg/g)較SF組(121.90與47.43 μg/g)高。對血管升壓素轉換酶(ACE)的抑制活性方面，SFH組之IC50 值為0.47 mg powder/mL較SF組的1.24 mg powder/mL為低。乳清粉末經腸胃道消化酵素水解後，SF與SFH組的胜肽分別由32.5及373.5 mg/g 增加為72.2及469.1 mg/g 。抑制ACE之IC50值方面，SF組由1.24上升至1.33 mg powder/mL，而SFH組之IC50值由0.47下降至0.41 mg powder/mL。SF組之胜肽分子量分佈範圍介於1520至380 Da之間，而SFH組之乳清胜肽分子量分布在1520-400 Da之間，兩組皆可得到四個主要的波峰區域，其中以SFH組之分子量450-400 Da之劃分物對抑制ACE能力最強，其有效抑制百分比(IER)為500%/mg/mL。將此劃分物依離子強度進行分離，可得到兩個波峰(B4-1與B4-2)物質，其IER分別可提高至2162與1307%/mg/mL，並以帶正電荷較弱之劃分物其有效抑制百分比(IER)較高。

Abstract

Soymilk was fermented with mixed lactic acid bacteria for up to 20h at 42℃. The whey was separated from the fermented soymilk and then freeze-dried. The effect of fermentation method on inhibitory activity against angiotensin I converting enzyme (ACE) and bioactive substances of whey powder were investigated. As the fermentation time extended to 20h, pH decreased from 6.55 to 4.41 and 4.15, acidity increased from 0.06 to 0.23 and 0.91%, respectively, for alone fermentation (SF) and combined fermentation (SFH). Soluble protein content of SFH increased from 62.6 to 236.7 mg/g that was six times higher than SF. Peptide content of SFH increased from 26.0 to 236.7 mg/g that was ten times higher than SF. γ-aminobutyric acid (GABA) were 136.1 and 201.5 mg/100g for SF and SHF, respectively. Daidzein and genistein of SFH (246.2 and 188.2 μg/g) was higher than that of SF (121.9 and 47.4μg/g). SFH showed the lower IC50 on ACE being 0.47 mg powder/mL. The SFH was further hydrolyzed by gastrointestinal proteases, IC50 decreased to 0.41 mg powder/ml. The molecular weight distribution of whey for SF was between 1520 –380 Da and for SFH was between 1520-400 Da by gel filtration using Sephadex G-15, and both showed four major fractions. A fraction of SFH had molecular weight ranged 450-400 Da showed the highest inhibitory efficiency ratio (IER) being 500 %/mg/mL. Further, this fraction was isolated by ion-exchange chromatography (SP-Sephadex C-25 column) to obtain two individual fraction with IER being 2162 (weaker positive charge) and 1307 %/mg/mL, respectively.

目錄
頁次
中文摘要……………………………………………………………….. i
英文摘要……………………………………………………………….. iii
目錄…………………………………………………………………….. v

表目錄………………………………………………………………….. xi
圖目錄………………………………………………………………….. xiii
一、前言………………………………………………………………… 1
二、文獻整理…………………………………………………………... 3
(一)、大豆之營養價值…..……..………………..……………….. 3
1.大豆蛋白質…………………………………….…............….. 3
2.異黃酮………………………………………………………... 4
(二)、乳酸菌…....………………………………………………… 5
1.乳酸菌的特性…..………………………………...………….. 5
2.乳酸菌對腸胃道的益處……………………………………... 6
3.影響乳酸菌生長的因素…………….……………………….. 7
(1)營養素…………………………………………………….. 7
(2)發酵溫度………………………………………………….. 7
4.乳酸菌之應用……………………………..……………...….. 8
(1)產生香味..……..……………...…………………………... 8
(2)去除異味及臭味………………….….…....……………… 8
(3)食品保存上之利用………………..…………………….... 9
(三)、發酵豆乳之特性……………………………………………. 9
1.生理活性物質的增加………..………………………………. 9
(1)胜肽……………………………………..…….………..…. 9
(2) γ-aminobutyric acid (GABA)…………………………….. 9
(3) 異黃酮…………………………………………………... 10
2.增進消化吸收性…….……………………………………….. 10
(1)蛋白質……………………………………..…………….. 10
(2)醣類…..………………………………………………..….. 11
(3)異黃酮…………………………………………………….. 11
(四)、影響發酵豆乳的因素…………………..………………….. 12
1.菌種….....…………………………………………………….. 12
2.不同添加物…………...…………………………………..….. 13
(五)、高血壓之定義及分類…...……………………….……....... 13
1.定義……....……....……………………..…….…....………… 13
2.分類……………………………….….……………….……… 14
(1)原發性高血壓………………….…………………………. 14
(2)續發性高血壓……..…………………………………….... 14
(六)、血管升壓素轉換酶(ACE)的生理活性……….….………… 15
1.ACE的生化特性及其降血壓的影響……..…………………. 15
2.抑制原理….……..……………………..…...….…....……….. 17
(七)、抑制高血壓之食品來源………………………………..….. 18
1.豆製品………..………………………………………………. 19
(1)醬油……………………….………………………………. 19
(2)納豆…………………………………….……………...….. 19
(3)大豆蛋白水解物………………….………………………. 20
2.乳製品…..….……..………………………...….…....……….. 20
3.魚類蛋白質………...………………..………...….…....…….. 21
(八)、抑制ACE活性胜肽之抑制類型…………………………… 22
1.抑制型…………….……….……….………………………… 22
2.前趨物型….………….………………...….…………………. 22
3.基質型………………….…….………………………………. 23
三、材料與方法……...….….................................................................. 24
(一)、實驗材料……….…………………………...………..…….. 24
(二)、實驗方法…….….……………………………...………....… 25
1.發酵豆乳之製備….....……………………................….…..... 25
2.酸度之測定…..………………………………………………. 26
3.可溶性蛋白質含量之測定………...……………...….…....… 26
4.胜肽含量之測定……..…….…………...….…....................... 27
5.游離胺基酸含量的測定……………………………………... 27
6.游離胺基酸組成分析…………............................................... 28
(1)萃取物之製備…………………………………………….. 28
(2)胺基酸之分析…………………………………………….. 28
7.異黃酮含量之測定…………………………………………... 29
8.發酵豆漿之乳清粉末對ACE抑制能力之測定…………... 29
9.IC50值的測定………………………………………..……… 30
10.消化試驗…………………………………………...….…..... 31
(1)乳清經腸胃道消化酵素水解之水解物製備…………….. 31
(2)乳清水解物對ACE抑制能力之測定…………………… 31
11.ACE抑制胜肽的純化與鑑定…………………….………. 31
(1)膠體過濾………………………………………………….. 31
(2)各劃分收集物對ACE抑制活性之測定………………… 32
(3)離子交換………………………………………………….. 32
(4)高效能液相層析………………………………………….. 33
12.統計分析………………………………………………..…... 34
四、結果與討論........................................................................................ 35
(一)、發酵方式對發酵豆乳成分之影響...................................... 35
1.pH值與酸度………………………………………………….. 35
2.可溶性蛋白質..…………...………….……………...….…..... 35
3.胜肽……..……………………………………………………. 36
4.游離胺基酸……….……………...…….…....……………….. 36
5.游離胺基酸組成分析………………………………………... 36
6.γ-aminobutyric acid (GABA)含量………...…......................... 37
7.異黃酮………………………………………………………... 37
(二)、發酵豆乳乳清粉末對ACE之抑制能力…….……………. 38
(三)、發酵豆乳乳清粉末經消化酵素水解後之胜肽含量與其抑
制ACE活性之影響………………………………………...
39
1.胜肽含量的變化…………………………………..…………. 39
2.對ACE的抑制……………………..………………………… 39
(四)、發酵豆乳乳清成分之管柱層析……………………….…… 40
1.G-15膠體層析………………………………………………... 40
(1)分子量分布及各劃分物對ACE抑制能力的影響………. 40
(2)HPLC分離………………………………………………... 41
2.SP-C25離子交換樹酯層析……………………….…………. 41
(1)離子強度之分離………………………………………….. 41
(2) HPLC分離……………………………………………….. 42
(3)劃分物對ACE抑制能力之影響…………………………. 42
五、結論………………………………………………………………... 43
六、參考文獻…………………………………………………………... 44
七、表…………………………………………………………………... 57
八、圖………………………………………………………………...… 69








表目錄
頁次
表一、發酵方式對發酵豆乳乳清之pH值與酸度的影響…………… 57
表二、發酵方式對發酵豆乳乳清粉末之可溶性蛋白質、胜肽與游離胺基酸含量的影響……………………………………………..
58
表三、發酵方式對發酵豆乳乳清粉末的游離胺基酸含量的影響…... 59
表四、發酵方式對發酵豆乳乳清粉末的GABA含量的影響….……. 60
表五、發酵方式與溶劑萃取對發酵豆乳乳清粉末中Daidzein 的影響…………………………………………………………..
61
表六、發酵方式與溶劑萃取對發酵豆乳乳清粉末中Genistein 的影響..........................................................................................
62
表七、發酵豆乳乳清粉末對ACE之IC50的影響…………………… 63
表八、腸胃道消化酵素水解對不同發酵方式之發酵豆乳乳清粉末胜肽含量的影響…………………………………………………..
64
表九、腸胃道消化酵素水解對不同發酵方式之發酵豆乳乳清粉末 IC50的影響………………………………………………………
65
表十、單獨發酵豆乳乳清粉末劃分收集物之ACE有效抑制百分比 66

表十一、混合發酵豆乳乳清粉末劃分收集物之ACE有效抑制百分比…………………………………………….……………….
67
表十二、混合發酵(SFH)之劃分物(B4)經離子交換後之劃分物的ACE有效抑制百分比………………………………………..
68














圖目錄
頁次
圖一、單獨發酵豆乳乳清粉末之膠體層析圖譜……………………… 69
圖二、混合發酵豆乳乳清粉末之膠體層析圖譜……………………… 70
圖三、混合發酵豆乳乳清經Sephadex G-15膠過濾後(Fig.2)收集波
峰B4的高效液相層析圖譜…………………………….............
71
圖四、混合發酵豆乳乳清(SFH)之劃分物(B4)的離子交換圖譜…….. 72
圖五、混合發酵豆乳乳清(SFH)之劃分物(B4)經離子交換後之劃分
物(B4-1)的高效液相層析圖譜......……………………………..
73
圖六、混合發酵豆乳乳清(SFH)之劃分物(B4)經離子交換後之劃分物(B4-2)的高效液相層析圖譜..............………………………..
74

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