臺灣博碩士論文加值系統

本實驗探討不同去乙醯度（dd 50, dd 75, dd 85, dd99）、濃度（0.5 ﹪, 1.0 ﹪, 1.5 ﹪）或pH值（3, 5）之幾丁聚醣與肌原纖維蛋白質混合加熱膠凝時，幾丁聚醣對肌原纖維蛋白質熱凝膠之凝膠強度與蛋白質變化的影響。由動態流變測定發現豬里肌肉中鹽溶性蛋白質（Salt-soluble proteins；SSP）的熱凝膠過程可分為低溫區（50 ℃以下）與高溫區（50 ℃以上）兩階段。添加幾丁聚醣於SSP中在低溫區並無明顯的流變特性變化，然而幾丁聚醣的添加卻會降低SSP在高溫區熱凝膠之複合模量（complex modulus；G*），可知幾丁聚醣胺基帶正電荷的特性干擾SSP的膠凝作用。無論去乙醯度為何，添加幾丁聚醣的濃度愈高，幾丁聚醣對SSP凝膠作用之干擾則愈強，造成幾丁聚醣/SSP混合系統熱凝膠之G*愈低。幾丁聚醣/SSP熱凝膠之tanδ（相角正切值）隨著加熱溫度的增加而降低，當加熱終點（75 ℃）時，幾丁聚醣/SSP熱凝膠之tanδ較單獨SSP熱凝膠之tanδ為高。而添加高濃度（1.5 ﹪）之dd 99幾丁聚醣，則可能因其胺乙醯基最少，以致於與SSP因離子鍵作用，而有最低之tanδ。
調整幾丁聚醣溶液pH至3時，黏度皆較未調整pH值時增加。當添加幾丁聚醣（pH 3）的濃度愈高，幾丁聚醣/SSP混合系統熱凝膠之G*愈高，亦即膠體彈性增高，因幾丁聚醣與SSP產生離子鍵結增加，故增強SSP在低溫區的膠凝作用。而dd 99因胺基正電荷數量增加，與SSP產生較強的離子鍵，故凝膠體之彈性現象較強。當幾丁聚醣溶液之pH值調整至5，其黏度則較未調整pH時為低。於相同去乙醯度時，添加幾丁聚醣（pH 5）的濃度愈高，幾丁聚醣/SSP混合系統熱凝膠之G*則愈低。而dd 99因正電荷數減少，在高溫區段與SSP形成氫鍵增加而干擾了SSP熱凝膠，故其G*下降。
幾丁聚醣/SSP混合系統之凝膠體以8 M urea處理後，進行SDS-PAGE膠體電泳，結果發現在焦集膠體部分有大分子蛋白質（＞200 KDa）的聚集，顯示幾丁聚醣與SSP形成新的共價鍵結而產生大分子聚合物。dd 50幾丁聚醣與SSP作用形成幾丁聚醣/SSP聚合物，導致凝膠體之肌凝蛋白重鏈（200 KDa）及肌動蛋白（45 KDa）的蛋白質帶有消失的現象。而pH 3及pH 5幾丁聚醣/SSP凝膠體之電泳結果則發現，因為幾丁聚醣胺基帶正電荷的改變，SSP凝膠體在電泳圖中大分子蛋白質聚合物的含量則較未調整pH前有減少的現象。

Rheological and thermal gelation characteristics of different degree of deacetylation (dd 50, dd 75, dd85, dd 99), concentration (0.5%, 1.0%, 1.5%) or pH (3, 5) of chitosan solution (in 1% lactic acid) and the mixture of chitosan and myofibrillar proteins were evaluated. Interactions of chitosan with myofibrillar proteins through SDS-PAGE during heating were also demonstrated. Thermal gelation processes of porcine salt-soluble proteins (SSP) extracted from tenderloin could be separated into the low temperature zone (< 50℃) and high temperature zone (> 50℃). No apparent rheological changes were noticed for chitosan/SSP mixture in the low temperature zone, but incorporation of chitosan reduced complex moduli (G*) of SSP in the high temperature zone. This result suggested that SSP thermal gelation be possibly interfered by the positive charges of amino group of chitosan molecule. Regardless of degree of deacetylation, increasing chitosan concentration caused stronger interference on SSP gelation and lowered G*. The tan δ (loss tangent) of chitosan/SSP gel decreased with increasing heating temperature. At the end of heating cycle (75℃), tan δ of chitosan/SSP gel was higher than that of SSP alone. Higher level (1.5%) of dd 99 chitosan, low in amount of acetyl amino group, had the lowest G* which possibly resulted from the ionic interaction with SSP.
Increased viscosity was noted for chitosan solution adjusted to pH 3 comparing with chitosan solution prior to pH adjustment. Higher chitosan concentration (at pH 3) resulted in higher G* or gel elasticity of chitosan/SSP mixture. This could be probably attributed to increased ionic interaction between chitosan and SSP, thus enhanced thermal gelation in low temperature zone. Therefore, higher numbers of positive charge for dd 99 chitosan (at pH 3) was shown to have stronger ionic interaction and gel elasticity. Viscosity of chitosan solution at pH 5 was shown to be lower than that of chitosan solution before adjusting pH. Regarding the same degree of deacetylation, increased chitosan level (at pH 5) reduced G* of chitosan/SSP mixture. Contrast to chitosan solution at pH 3, dd 99 possessed less amounts of positive charge which interfered SSP thermal gelation at high temperature zone through the formation of hydrogen bonding.
SDS-PAGE results from chitosan/SSP gel following 8M urea treatment revealed large protein molecule (> 200 KDa) aggregation in the stacking gel, which indicated formation of new covalent linkages between chitosan and SSP into chitosan/SSP polymer. Electrophretogram from dd 50 also showed disappearing myosin heavy chain (MW 200 KDa) and actin (MW 45 KDa) protein bands that resulted from formation of chitosan/SSP polymer. Intensities or concentration of large chitosan/protein polymers for chitosan solution at either pH 3 or 5 were less, due to changes in the numbers of positive charge, than that of chitosan solution before pH adjustment.
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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
第一章前 言…………………………………………………………1
第二章文獻回顧………………………………………………………3
一、幾丁質及幾丁聚醣之來源、分佈、化學構造及應用………3
（一）幾丁質（chitin）………………………………………3
（二）幾丁聚醣（chitosan）…………………………………3
二、幾丁質及幾丁聚醣之理化性質……………………………6
（一）幾丁質…………………………………………………..6
（二）幾丁聚醣………………………………………………..7
（三）聚陽離子性質（polycationic properties）…………….8
三、幾丁質與幾丁聚醣之製備…………………………………...8
（一）幾丁質之製備…………………………………………..8
1.鈣鹽之去除……………………………………………….8
2.蛋白質之去除…………………………………………….8
（二）幾丁聚醣之製備………………………………………10
四、幾丁質與幾丁聚醣的應用………………………………….10
（一）廢水處理………………………………………………10
（二）生化方面………………………………………………13
（三）食品方面………………………………………………13
1.抗菌作用…………………………………………..13
2.膳食纖維…………………………………………..13
3.保水與乳化作用…………………………………..16
4.香味料……………………………………………..16
5.抗氧化作用………………………………………..16
6.其他食品上的應用………………………………..17
五、蛋白質的凝膠機制………………………………………….17
六、鹽溶性肌原纖維蛋白質的熱凝膠特性…………………….19
七、溫度和pH值對鹽溶性蛋白質熱凝膠的影響……………...21
八、多醣類混合系統之凝膠機制……………………………….22
九、添加多醣類膠體對鹽溶性蛋白質熱凝膠的影響………….24
第三章研究目的………………………………………………………29
第四章材料與方法……………………………………………………30
一、實驗材料…………………………………………………….30
二、實驗方法…………………………………………………….30
（一）幾丁聚醣溶液之製備…………………………………30
（二）快速黏度之測定………………………………………31
（三）豬肉鹽溶性蛋白質的萃取……………………………31
（四）蛋白質含量測定………………………………………32
（五）動態流變測定…………………………………………32
（六）十二基硫酸鈉-聚丙烯醯胺膠體電泳
（SDS-PAGE）……………………………………………34
1.試藥配置………………………………………….34
（1）Lower buffer（分離膠體緩衝液（separating
gel buffer））…………………………………34
（2）Upper buffer（焦集膠體緩衝液（stacking
gel buffer））…………………………………34
（3）丙烯醯胺液……………………………………34
（4）電泳槽緩衝液（0.192 M Glycine）………35
（5）樣品處理液（sample buffer）………………35
（6）10 ﹪過硫酸銨溶液（ammonium persulfate
；APS）…………………………………….35
（7）蛋白質分子量標準品…………………………35
（8）0.2 ﹪蛋白質染色液（staining solution）…….35
（9）脫色液（destaining solution）……………….35
（10）7.5 ﹪醋酸溶液……………….…………….36
2.鑄膠操作…………………………………………..36
3.樣品處理…………………………………………..36
4.電泳操作…………………………………………..37
第五章結果與討論……………………………………………………40
一、去乙醯度對幾丁聚醣溶液的影響………………………….40
（一）pH值…………………………………………………...40
（二）黏度……………………………………………………40
二、幾丁聚醣與肌原纖維蛋白質混合系統之流變特性……….44
（一）肌原纖維蛋白質之加熱凝膠特性……………………44
（二）幾丁聚醣與肌原纖維蛋白質混合系統的凝膠特性…46
三、以SDS-PAGE觀察幾丁聚醣對肌原纖維蛋白質加熱凝
膠的影響……………………………………………………53
四、不同pH值之幾丁聚醣與肌原纖維蛋白質混合系統之
流變特性……………………………………………………58
（一）黏度……………………………………………………58
（二）pH 3之幾丁聚醣與肌原纖維蛋白質混合系統的凝
膠特性…………………………………………………64
（三）pH 5之幾丁聚醣與肌原纖維蛋白質混合系統的凝
膠特性…………………………………………………69
五、以SDS-PAGE觀察不同pH值之幾丁聚醣對肌原纖維
蛋白質加熱凝膠的影響……………………………………74
（一）pH 3之幾丁聚醣對肌原纖維蛋白質的影響…………74
（二）pH 5之幾丁聚醣對肌原纖維蛋白質的影響…………78
第六章結 論…………………………………………………………85
第七章參考文獻………………………………………………………87

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