臺灣博碩士論文加值系統

本實驗首先探討寡木糖（xylooligosaccharides）、海藻糖（trehalose）和山梨糖醇（sorbitol）添加於豬里肌肉鹽溶性蛋白質中，經加熱所製成鹽溶性/糖類蛋白質混合膠（SSP / sugar mixed gels）之膠強度、可擠壓水分、膠體電泳和蛋白質表面疏水性；其次將SSP分別混合寡木糖、海藻糖、山梨糖醇和蔗糖，加熱凝膠後，於-20 ℃下凍藏，分別在0、4、8、12和20週取樣分析。結果顯示，不論加熱溫度為71.1 ℃或76.7 ℃，當蛋白質濃度為40 mg/mL時混合膠膠體呈現弱膠，而蛋白質濃度為60 mg/mL時，混合膠之膠強度隨著添加寡木糖增加而增加，以添加6%寡木糖的膠強度（2.99 ，N × cm）最高，在蛋白質濃度為120 mg/mL時，添加4%和8%寡木糖時其混合膠之膠強度達最大。添加三種糖類皆可造成肌原纖維蛋白質混合膠體的可擠壓水分（expressible moisture, EM）降低，亦即膠體保水性增加。隨著添加糖濃度（4-12%）增加而混合膠體保水性也提高，其中又以添加12%海藻糖的EM值最低。電泳結果顯示加熱後的蛋白質帶較未加熱者少，顯示蛋白質在加熱後已變性沉澱；經加熱後，添加寡木糖之蛋白質帶的強度較未添加對照組低。在未加熱及加熱71.1 ℃下，添加寡木糖者其蛋白質相對表面疏水性（relative surface hydrophobicity, RSo）較添加海藻糖和山梨糖醇低，且RSo也隨著寡木糖的添加濃度增加而降低，RSo值越低，表示蛋白質的疏水性區域暴露較少，亦即可能蛋白質變性程度較低。
糖類/SSP混合膠於冷凍儲藏期間結果顯示，隨著凍藏期間的增加，混合膠的冷凍/解凍安定性和蛋白質溶解度有下降趨勢，添加糖類試驗組則較未添加糖對照組有較佳之冷凍/解凍安定性和蛋白質溶解度。隨著添加糖濃度增加混合膠的冷凍/解凍安定性和蛋白質溶解度亦顯著增加，而以添加寡木糖有最佳之冷凍/解凍安定性和蛋白質溶解度。 SDS-PAGE結果顯示蛋白質帶的強度隨著儲藏週期增加逐漸有降低的趨勢，顯示蛋白質濃度有降低的現象，而添加寡木糖試驗組發現經12週凍藏後蛋白質強度幾乎消失，顯示蛋白質濃度越來越低。隨著儲藏週期增加，各組之蛋白質相對表面疏水性有逐漸上升之趨勢，顯示凍藏期間增加，造成蛋白質的疏水性區域暴露越多，糖類試驗組中以添加寡木糖有較低之蛋白質相對表面疏水性。綜合以上分析結果，添加糖於冷凍肉製品中具有良好的抗凍效果，尤以寡木糖的效果最佳。

The gel strength, expressible moisture, SDS-PAGE and protein surface hydrophobicity of the porcine SSP/sugar mixed gels were investigated. Furthermore, the physical and chemical properties of heated SSP/sugar mixed gels under 20 weeks of frozen storage were evaluated. Results indicated, regardless heating temperatures, weak SSP/sugar mixed gels at protein concentration of 40 mg/mL. At protein concentration of 60 mg/mL, the gel strength of SSP/sugar mixed gels increased with increasing sugar levels. Addition of sugars lowered expressible moisture and thus elevated the water-holding capacity (WHC) of mixed gels. Increase in sugar levels resulted in increasing WHC. Results from SDS-PAGE revealed severe protein denaturation following heating of mixed gels. The protein surface hydrophobicity (RSo) of xylooligosaccharides-added treatment was lower than other treatments. The low RSo provides an indication of possible less exposure of protein hydrophobic groups.
Results from freeze/thaw stability and protein solubility showed a descending pattern of SSP/sugar mixed gels following frozen storage, with xylooligosaccharides-added treatment being the highest among treatments. Increasing sugar levels improved freeze/thaw stability and protein solubility of frozen mixed gels. Accordingly, the intensity of protein bands from SDS-PAGE gradually decreased with frozen periods. The RSo of frozen mixed gels gradually increased with frozen periods implying possible higher degree of protein denaturation. Addition of xylooligosaccharides resulted in the lowest RSo among treatments.
In conclusion, incorporation of sugar (sorbitol, sucrose, xylooligosaccharides) to frozen meat products asserts a cryoprotective function, and xylooligosaccharides appears to be better than other sugars.

目 錄
頁次
中文摘要……………………………………………………………...... I
英文摘要……………………………………………………………...... III
謝誌…………………………………………………………………….. V
目錄…………………………………………………………………...... VI
表目錄………………………………………………………………...... X
圖目錄………………………………………………………………...... XI
表附錄………………………………………………………………….. XIV
第一章 前 言………………………………………………………...... 1
第二章 文獻回顧……………………………………………………… 3
一、寡糖…………………………………………………………… 3
（一）寡糖之簡介………………………………………………… 3
（二）寡糖之特性………………………………………………… 5
（三）寡木糖（xylooligosaccharides）………………………….. 5
1.寡木糖之介紹………………………………………………... 5
2.寡木糖之理化特性…………………………………………... 5
3.寡糖之生理特性……………………………………………... 9
二、海藻糖（trehalose）………………………………………….. 16
（一）海藻糖之簡介……………………………………………… 16
（二）海藻糖之理化特性………………………………………… 17
（三）海藻糖的應用……………………………………………… 18
三、山梨糖醇（sorbitol）………………………………………… 22
四、蔗糖（sucrose）……………………………………………… 22
（一）蔗糖之物化性質…………………………………………… 22
五、肌肉蛋白質…………………………………………………… 25
1.肌漿蛋白質（sacroplasmic proteins）……………………… 25
2.不溶性基質蛋白質（stromal proteins）…………………… 26
3.肌原纖維蛋白質（myofibrillar proteins）…………………. 26
（一）肌原纖維蛋白質的熱凝膠特性…………………………... 26
六、蛋白質的熱凝膠機制………………………………………… 28
七、蛋白質表面疏水性…………………………………………… 29
八、糖類對蛋白質加熱凝膠之影響……………………………… 31
九、抗凍劑對蛋白質冷凍變性之影響…………………………… 32
第三章 研究目的……………………………………………………… 35
第四章 材料與方法…………………………………………………… 36
一、實驗材料…………………………………………………........ 36
1.原料肉..................................................36
2.化學藥品……………………………………………………... 36
二、實驗方法……………………………………………………… 37
第一部分：鹽溶性蛋白質與不同糖類混合膠之物性…………… 37
（一）鹽溶性蛋白質（salt-soluble proteins, SSP）萃取………… 37
1.緩衝溶液配製………………………………………………..... 37
(1) 0.1 M NaCl/0.05 M sodium phosphate buffer, pH 7.0…….37
(2) 0.6 M NaCl/0.05 M sodium phosphate buffer, pH 7.0…….37
(3) 2.4 M NaCl/0.2 M sodium phosphate buffer, pH 7.0……..37
2.豬里肌肉鹽溶性蛋白質之製備……………………………..... 37
（二）蛋白質濃度測定…………………………………………… 38
1.蛋白質濃度校正曲線………………………………………..... 38
2.蛋白質濃度測定……………………………………………..... 38
（三）膠強度……………………………………………………… 39
1.鹽溶性蛋白質混合膠之製備………………………………..... 39
2.膠強度之測定………………………………………………..... 39
（四）蛋白質表面疏水性…………………………………………. 39
1.螢光探針(8-anilino-1-naphthalene-sulfonic acid, ANSA)溶液之配
製…………………………………………………....................... 39
2.蛋白質表面疏水性測定……………………………………..... 39
（五）十二基硫酸鈉-聚丙醯烯胺膠體電泳(SDS-PAGE)………. 40
1.試藥配製………………………………………………………. 40
(1)Lower buffer（分離膠體緩衝液（separating gel buffer））
……………………………………………………………..................40
(2)Upper buffer（焦集膠體緩衝液（stacking gel buffer））
…………………………………………................................40
(3)丙烯醯胺液…………………………………………………. 41
(4)電泳槽緩衝液（0.192 M Glycine）……………………….. 41
(5)樣品處理液（sample buffer）……………………………… 41
(6) 10%過硫酸銨溶液（ammonium persulfate；APS）……… 41
(7)蛋白質分子量標準品………………………………………. 41
(8) 0.2%蛋白質染色液（staining solution）…………………… 41
(9)脫色液（destaining solution）……………………………… 41
(10) 7.5%醋酸溶液…………………………………………….. 42
2.鑄膠操作………………………………………………………. 42
3.樣品處理………………………………………………………. 42
(1)蛋白質標準品………………………………………………. 42
(2)樣品…………………………………………………………. 42
4.電泳操作………………………………………………………. 43
（六）可擠壓之水分……………………………………………… 44
（七）統計分析…………………………………………………… 44
第二部分：鹽溶性蛋白質與不同糖類混合之冷凍儲藏安定性
……………………………………………………………………… 44
（一）蛋白質溶解度……………………………………………… 45
（二）冷凍/解凍安定性…………………………………………… 45
（三）統計分析…………………………………………………… 46
第五章 結果與討論…………………………………………………… 47
第一部分：肌原纖維蛋白質與不同糖類混合膠之物性………… 47
（一）膠強度……………………………………………………… 47
（二）保水性……………………………………………………… 50
（三） 以SDS-PAGE觀察糖類對肌原纖維蛋白質之影響……… 52
（四）糖類對肌原纖維蛋白質相對表面疏水性之影響………… 56
第二部分：肌原纖維蛋白質與糖類混合膠的冷凍儲藏安定性……………………………………………………......................59
（一）冷凍解凍安定性……………………………………………. 59
（二）蛋白質溶解度……………………………………………… 62
（三）冷凍儲存期間糖類對肌原纖維蛋白質之影響…………… 64
（四）冷凍儲存週期間糖類對肌原纖維蛋白質相對表面疏水性
之影響……………………………………………………..................75
第六章 結論…………………………………………………………… 82
第七章 參考文獻……………………………………………………… 83
第八章 附錄………………………………………………………….... 95


表目錄
頁次
表一 各種寡醣產品之種類、結構及來源……………………… 4
表二 海藻糖在食品上之應用…………………………………… 20
表三 海藻糖之性質……………………………………………… 21
表四 不同濃度寡木糖添加於不同濃度之鹽溶性蛋白質中於71.1 °C下加熱凝膠之膠體其膠強度影響………………........................................50
表五 不同濃度寡木糖添加於不同濃度之鹽溶性蛋白質中於76.7 °C下加熱凝膠之膠體其膠強度影響……………..........................................51
表六 不同濃度之糖類（寡木醣、海藻糖、山梨糖醇）與鹽溶性蛋白質混合膠體之保水性………………………………......................................53
表七 冷凍儲藏期間SSP/糖類混合膠冷凍/解凍離水率之變化.. 61
表八 冷凍儲藏期間SSP/糖類混合膠蛋白質溶解度之變化…… 63



圖目錄
頁次
圖一 半纖維素之主要成分………………………………….. 6
圖二 大豆寡醣與寡木醣之製程…………………………….. 7
圖三 寡木醣之結構………………………………………….. 8
圖四 寡木醣於不同溫度下之黏度………………………….. 10
圖五 寡木醣之熱穩定性…………………………………….. 11
圖六 寡木醣之酸穩定性…………………………………….. 12
圖七 寡木醣之貯藏安定性………………………………….. 13
圖八 寡木糖的應用………………………………………….. 14
圖九 α,α-海藻糖之結構……………………………………... 19
圖十 山梨糖醇之結構式…………………………………….. 23
圖十一 蔗糖之結構式………………………………………….. 24
圖十二 肌凝蛋白的分子模式圖……………………………….. 27
圖十三 濃度4, 8, 12%之寡木糖與SSP混合凝膠的電泳膠片圖………………………………………………..........................53
圖十四 濃度4, 8, 12%之海藻糖與SSP混合凝膠的電泳膠片圖………………………………………………..........................54
圖十五 濃度4, 8, 12%之山梨糖醇與SSP混合凝膠的電泳膠片圖……………………………………………............................55
圖十六 SSP與不同濃度之糖類在未加熱下其相對表面疏水性圖……………………………………………………......................57
圖十七 SSP與不同濃度之糖類在加熱下（71.1 ℃）其相對表面疏水性圖……………………………………………............................58
圖十八 濃度4, 8, 12%之寡木糖和海藻糖與SSP混合凝膠於-20 ℃下冷凍貯藏0週之電泳膠片圖………………..............................................65
圖十九 濃度4, 8, 12%之山梨糖醇和蔗糖與SSP混合凝膠於-20 ℃下冷凍貯藏0週之電泳膠片圖………………..............................................66
圖二十 濃度4, 8, 12%之寡木糖和海藻糖與SSP混合凝膠於-20 ℃下冷凍貯藏4週之電泳膠片圖………………..............................................67
圖二十一 濃度4, 8, 12%之山梨糖醇和蔗糖與SSP混合凝膠於-20 ℃下冷凍貯藏4週之電泳膠片圖………………..............................................68
圖二十二 濃度4, 8, 12%之寡木糖和海藻糖與SSP混合凝膠於-20 ℃下冷凍貯藏8週之電泳膠片圖………………..............................................69
圖二十三 濃度4, 8, 12%之山梨糖醇和蔗糖與SSP混合凝膠於-20 ℃下冷凍貯藏8週之電泳膠片圖………………..............................................70
圖二十四 濃度4, 8, 12%之寡木糖和海藻糖與SSP混合凝膠於-20 ℃下冷凍貯藏12週之電泳膠片圖……………… ..............................................71
圖二十五 濃度4, 8, 12%之山梨糖醇和蔗糖與SSP混合凝膠於-20 ℃下冷凍貯藏12週之電泳膠片圖……………… ..............................................72
圖二十六 濃度4, 8, 12%之寡木糖和海藻糖與SSP混合凝膠於-20 ℃下冷凍貯藏20週之電泳膠片圖……………… ..............................................73
圖二十七 濃度4, 8, 12%之山梨糖醇和蔗糖與SSP混合凝膠於-20 ℃下冷凍貯藏20週之電泳膠片圖……………… ..............................................74
圖二十八 SSP與不同濃度之糖類在-20 ℃下凍藏0週的相對表面疏水性曲線圖………………………………………................................76
圖二十九 SSP與不同濃度之糖類在-20 ℃下凍藏4週的相對表面疏水性曲線圖………………………………………................................77
圖三十 SSP與不同濃度之糖類在-20 ℃下凍藏8週的相對表面疏水性曲線圖………………………………………................................78
圖三十一 SSP與不同濃度之糖類）在-20 ℃下凍藏12週的相對表面疏水性曲線圖…………………………………....................................79
圖三十二 SSP與不同濃度之糖類在-20 ℃下凍藏20週的相對表面疏水性曲線圖……………………………………..................................80



表附錄
頁次
表一 未加熱糖類（4%）對鹽溶性肌原纖維蛋白質相對表面疏水性之螢光吸光值………………………………………............................................95
表二 未加熱糖類（8%）對鹽溶性肌原纖維蛋白質相對表面疏水性之螢光吸光值…………………………………………..........................................96
表三 未加熱糖類（12%）對鹽溶性肌原纖維蛋白質相對表面疏水性之螢光吸光值……………………………………..............................................97
表四 加熱糖類（4%）對鹽溶性肌原纖維蛋白質相對表面疏水
性之螢光吸光值…………………………………………..............................98
表五 加熱糖類（8%）對鹽溶性肌原纖維蛋白質相對表面疏水
性之螢光吸光值…………………………………………..............................99
表六 加熱糖類（12%）對鹽溶性肌原纖維蛋白質相對表面疏
水性之螢光吸光值…………………………………………............................100
表七 糖類與肌原纖維蛋白質混合於-20 ℃下儲藏0週時的表
面疏水性之螢光吸光值……………………………………............................101
表八 糖類與肌原纖維蛋白質混合於-20 ℃下儲藏4週時的表
面疏水性之螢光吸光值……………………………………............................102
表九 糖類與肌原纖維蛋白質混合於-20 ℃下儲藏8週時的表
面疏水性之螢光吸光值…………………………………..............................103
表十 糖類與肌原纖維蛋白質混合於-20 ℃下儲藏12週時的表
面疏水性之螢光吸光值……………………………………............................104
表十一 糖類與肌原纖維蛋白質混合於-20 ℃下儲藏20週時的表面疏水性之螢光吸光值……………………………………..............................................105

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