臺灣博碩士論文加值系統

由鯖魚﹙Scomber australasicus﹚頭部表面分離出一株冰核菌，編號MACK-4，經鑑定為Pseudomonas fluorescens。培養於含2.5% glycerol之nutrient broth (NB-G)時最適生長條件為15oC及pH 6.5，培養54 h可得到最高冰核活性﹙ice-nucleating activity, INA﹚，但以25 oC以上溫度培養48 h 即喪失活性。P. fluorescens MACK-4以peptone 為氮源及碳源以dextrin或starch培養時，菌數成長最迅速，而sorbitol、 mannose及starch為碳源之培養可得到最高冰核活性。以sorbitol為碳源，氮源為無機氮鹽、malt extract 或 gelatine時， 菌生長不易，但氮源為peptone、 yeast extract、 tryptone 或skim milk則生長迅速。於30C 培養後可以5C低溫誘導出活性，飢餓培養基則無法促進活性表現。由化學試劑與金屬離子作用結果，推測冰核活性物質之蛋白質部分含SH基團。P. fluorescens MACK-4之穩定期培養液pH約為7.2- 7.5，菌濾液之冰核活性log(INA/mL)為0.710，菌液以1 M 鹽酸調整至pH 6.0，離心過濾除菌後，可得最高濾液活性1.95。不同體積純水，加入菌液或粗萃取冰核活性物質（extracellular ice nucleation matter, EINM）進行冷凍，可減少過冷卻現象，且體積愈大者效果愈明顯，但卻無助於減少完全冷凍所需時間。添加不同量菌液於純水，菌量增加，減少過冷卻之程度愈明顯，但同樣無法減少完全冷凍所需時間。牛奶樣品添加菌液與EINM可減少過冷卻現象，且添加EINM較未添加者完全凍結所需時間短，10% starch結果與純水系統相同，但添加於果汁與魚漿其冷凍曲線則不因添加而有所改善。推論冰核菌主要為促進初始冰核形成，無助於冰核成長。添加不同濃度抗凍劑，當濃度達20%，菌液與EINM均無活性，glycerol會明顯抑制活性，其他糖類之影響依濃度增加而增加，且EINM較菌液易受影響。去除P. fluorescens MACK-4菌體後濾液之冰核活性高於細胞壁膜物質與胞內物質，濾液藉50 kDa 濃縮過濾膜分離冰核活性物質(EINM)。IAA、-Me、SDS、urea和金屬離子會抑制所分離出之EINM活性，因此EINM內蛋白質可能具SH官能基，Protease可水解EINM。電泳分析結果顯示該EINM包含兩個分子量大於670 kDa之醣蛋白及一個約550 kDa之蛋白質所組合而成的複合體。兩個分子量大於670 kDa之醣蛋白可能是由三個以上且分子量在140∼180 kDa間之蛋白質與醣結合而成之醣蛋白，該醣蛋白會因SDS處理而分離為蛋白質與醣基，說明該醣蛋白之蛋白質與醣基是以疏水性鍵結或親水性鍵結結合而成。

An ice-nucleating bacterium, designated as MACK-4, was isolated from ice-stored mackerel (Scomber australasicus) and identified as a Pseudomonas fluorescens. The optimal temperature and pH for its growth in nutrient broth with 2.5% glycerol (NB-G) were 15C and 6.5, respectively. The maximal ice nucleating activity (INA) was obtained after 54 h incubation at 15C. However, the INA almost completely lost after 48 h incubation at 25C or higher temperatures. The highest ice-nucleating activity was observed on those grown in the media with sorbitol, mannose or starch using peptone as nitrogen source. According to OD600, the cells seemed to be difficult to grow in media with inorganic substances, malt extract or gelatine nitrogen sources. However, rapid growth was observed on the media with peptone, yeast extract, tryptone or skim milk. The concentrations of carbon or nitrogen sources did not significant affect the INA. The INA was greatly induced when the growth temperature was shifted from 30 to 5C. However, it was not affected by transferring the cells into the media with deficiency of nutrients. According the chemical agents and metal effects on INA, the active site on the proteins of INA components might contain SH group. The pH of culture at stationary stage was 7.2-7.5, while the INA was 0.710. The maximum ice nucleation activity of extracellular ice nucleation matter (EINM) was 1.95 when the pH of culture was adjusted to 6.0. The temperature of supercooling point was raised during freezing, when the water, milk or 10% starch was mixed with active bacteria cells or EINM. The decrease of supercooling point increased with the volume of water and the cells or EINM added. However, the addition of active bacteria cells or EINM did not affect the freezing time. No significant difference of the freezing curve between control and EINM or cells containing juice and surimi was observed. The cryoprotectants inactivated the INA of active bacteria cells or EINM, and in the freezing curve of lost when the concentration was over 20%. Glycerol and carbohydrates could inhibit, however, the degree of inhibition of carbohydrate increased with the increase of concentration. The extracellular ice nucleating matter (EINM) from P. fluorescens MACK-4 was isolated by using 50 kDa membrane fractionation. The ice nucleating activity (INA) of the isolated INM was inhibited by IAA, -Me, SDS, urea and divalent metal ions. This phenomenon suggested that the active site of the proteins on EINM might contain SH group. Protease could hydrolyze the proteins on EINM and consequently increased the INA of EINM. Electrophoretic analysis suggested that EINM was glycoprotein and easily de-glycosylated during isolation. The INA of MACK-4 was found to be highest in broth, then cell plasma and membrane. The EINM consisted of 2 glycoproteins with MW>670kDa and 1 protein with MW around 550 kDa. These glycoproteins were considered to be polysaccharides bond with at least 3 proteins with MW between 140 and 180 kDa. SDS could easily rupture the bounding of these glycoproteins, suggesting the bounds between polysaccharides and proteins be hydrophobic or hydrophilic interactions.

表次 Ⅳ
圖次 Ⅴ
中文摘要 Ⅵ
英文摘要 Ⅷ
研究動機與目的 Ⅹ
實驗設計 ⅩⅡ
第一章 文獻整理 1
一、 凍結之形成 1
A、冰核之形成 1
B、細菌冰核 2
二、 冰核菌之特性 3
A、冰核菌之分離與生長條件探討 3
B、冰核物質特性探討 5
三、 冰核菌活性物質之組成 7
A、冰核結構之蛋白質成分 8
B、冰核結構之sugars成分 9
C、冰核結構之phosphatidylinositol 11
D、多胺類對冰核結構之影響 12
　四、冰核菌與冰核物質之應用 14　
第二章 鯖魚體表冰核菌之分離、鑑定及其特性 18
　壹、摘要 18
　貳、前言 20　
　參、實驗材料 21
　　一、原料 21
二、培養基 21
三、實驗藥品 21
四、儀器 22
　肆、實驗方法 23
　　　一、培養 23
二、穿透式電子顯微鏡 24
　　　三、革蘭式染色 24
　　　四、API 20NE系統之鑑定 24
五、冰核活性測定 24
　　　六、最適培養條件條件與影響活性因子探討 25
　　　七、菌液活性影響因子探討 25
　伍、結果與討論 26
　　　一、冰核菌之分離 26
　　　二、探討MACK-4之培養條件及其對活性之影響 27
　　　三、最適培養之溫度與pH 28
　　　四、pH 與溫度對MACK-4之冰核活性影響 29
第三章 探討增進P. fluorescens MACK-4冰核活性之培養條件 31
壹、 摘要 31
貳、 前言 33
參、 材料與方法 34
一、準備菌種 34
二、金屬鹽類、碳與氮源之影響 34
三、冰核活性測定 34
四、化學試劑與金屬離子對冰核活性之影響 34
五、低溫處理與營養缺乏對冰核活性之誘導 35
六、統計分析 35
肆、 結果與討論 35
一、 碳與氮源對MACK-4之生長與活性影響 35
二、 培養基中碳與氮源濃度之影響 37
三、 低溫處理與營養缺乏對MACK-4之生長與活性影響 38
四、 金屬鹽類之影響 39
五、 金屬離子與化學試劑之影響 39
第四章P. fluorescens MACK-4 與其冰核活性物質在食品冷凍加工上
之應用 41
壹、 摘要 41
貳、 前言 43
參、 材料與方法 45
一、準備菌體 45
二、樣品製備 45
三、粗萃取冰核活性物質製備 45
四、冷凍曲線 46
五、冰核活性測定 46
六、抗凍劑對冰核活性之影響 46
肆、 結果與討論 46
一、 粗萃取液製備 46
二、 純水冷凍 47
三、 食品冷凍 47
四、 抗凍劑之影響 48
第五章 P. fluorescens MACK-4之冰核活性物質的分離與特性 50
壹、 摘要 50
貳、 前言 52
參、 材料與方法 53
一、 粗冰核活性物質之萃取 54
二、 膜物質分離 54
三、 超過濾濃縮 54
四、 醣基染色反應 54
五、 總醣含量分析 54
六、 酵素作用之影響 55
七、 冰核活性測定 55
八、 化學試劑與金屬離子對冰核活性之影響 55
九、電泳分析 55
肆、 結果與討論 56
一、 菌膜物質分離 56
二、 冰核活性物質分離 56
三、 金屬離子對冰核活性物質活性之影響 58
四、 化學試劑對冰核活性物質活性之影響 58
五、 酵素對冰核活性物質活性之影響 59
綜合結論 61
參考文獻 62

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