臺灣博碩士論文加值系統

本研究自食品工業發展研究所、土壤、水、稻草、市售日本納豆商品及其他納豆相關產品中，分離出Bacillus菌屬65株，利用血纖維平板試驗法進行菌株初步篩選，挑選出高活性納豆激酶生產菌，探討其最適固態與液態培養條件，以提高產量；之後分別將納豆激酶粗酵素液精製，並測定製品酵素活性回收率及穩定性之差異。結果顯示，以分離自市售日本納豆商品編號C13菌株，於37℃、48小時培養條件下其納豆激酶活性328.2 FU/g最高。將C13菌株於紫外線照射誘導突變試驗，分離出46株突變株，其中又以編號CM13突變株之納豆激酶活性421.2 FU/g最高。CM13突變株在固態培養試驗中，以黃豆為最適培養基質，接種量1.0mL (107~108 cell/mL)、相對濕度90%、在37℃培養32小時，其納豆激酶活性為469.2 FU/g。液態培養試驗中，經37℃培養32小時納豆激酶活性為70.3 FU/mL。固態培養納豆激酶粗酵素液在60℃及37℃穩定性試驗中，穩定性較液態培養高，故精製後回收率以固態培養63.0%高於液態培養39.8%，其主要原因為培養過程中納豆產生黏性物質聚麩胺酸 (γ-poly-glutamic acid, γ-PGA)，對於納豆激酶穩定性有提高的效果，而添加0.1% γ-PGA於液態培養粗酵素液，可明顯提高納豆激酶穩定性及製品酵素回收率。

In this study, 65 Bacillus spp. had been isolated from Bacillus stock of Food Industry Research And Development Institute, soil, water, rice straw, Japanese commercial natto and other natto products. First, plasminogen-free fibrin plate method was used to pick out the high nattokinase production strains from 65 Bacillus spp.. Second, the optimal condition for cultivation of solid state fermentation and submerged culture for Bacillus strains was estabalished to increase nattokinase production. Than, crude enzyme of nattokinase was purified and identified the different recoveries identified and stability of nattokinase activity from each strains examined. The results indicated that strain number C13 which was isolated from Japanese commercial natto produced the highest nattokinase activity, 328.2 FU/g at 37℃ from 48 hours of culture. Explore sure of C13 strain to ultraviolet rays induced mutation and 46 mutant strains were. Mutant CM13 could produced the highest nattokinase activity 421.2 FU/g. Soybean was the optimal medium for CM13 mutant solid state fermentation. The optimal cultivation condition for inoculum size, relative humidity, temperature and time were 1.0 mL (107~108 cell/mL), 90%, 37℃ and 32 hours, respectively The highest activity of nattokinase achieved was 469.2 FU/g. Using submerged culture, CM13 mutant gave the activity of nattokinase approximately 70.3 FU/mL when incubated at 37℃ for 32 hours. At 60℃ or 37℃ the nattokinase from solid substrate fermentation was more stable than from submerged culture. The recovery of nattokinase purified from solid substrate fermentation reached 63.0%, much higher than 39.8% from submerged culture. This is was because of the γ-poly-glutamic acid (γ-PGA) which produced from natto during the process of cultivation. γ-PGA increased the stability of nattokinase, and the recovery was also enhanced after purification. Adding 0.1% γ-PGA to in crude enzyme from submerged fermentation increased the stability and recovery of nattokinase sigificantly.

目 錄
目 錄 I
表 次 II
圖 次 III
中文摘要 IV
英文摘要 V

壹、前 言 1
貳、文獻整理 2
一、納豆 2
(一) 納豆的製程 2
(二) 納豆生理功效 2
(三) 納豆的特殊風味成分及黏性物質 4
二、聚麩胺酸 (γ-poly-glutamic acid, γ-PGA) 5
(一) γ-PGA之發現及特性 5
(二) γ-PGA之應用 5
1. 環境保護及廢水處理 6
2. 食品工業上之應用 6
3. 醫藥上之應用 6
4. 化妝品上之應用 7
5. 農業和綠化保護用途 7
四、納豆激酶 8
(一) 納豆激酶的發現 8
(二) 納豆激酶分解血栓機制 10
1. 血栓的形成 10
2. 分解血栓機制 10
(三) 納豆激酶製品之生產 12
1. 傳統固態發酵加工 12
2. 固態培養萃取分離精製 12
3. 液態發酵生產納豆激酶 13
五、微生物酵素生產 17
(一) 微生物酵素生產 17
1. 酵素生產菌的篩選 17
2. 突變菌株的篩選 18
(二) 微生物之培養 18
1. 固態培養 19
2. 液態培養 19
參、材料與方法 23
一、實驗架構 23
(一) 高活性納豆激酶生產菌之篩選 23
(二) 固態與液態培養之納豆激酶精製及其穩定性之探討 24
二、實驗材料 25
(一) 原料 25
(二) 菌株來源 25
(三) 試藥 25
1. 培養基 25
2. 藥品 26
(四) 儀器設備 27
三、實驗方法 28
(一) 高活性納豆激酶生產菌之篩選 28
1. 菌種分離與培養 28
(1) 自然環境分離納豆激酶生產菌 28
(2) 稻草分離納豆激酶生產菌 28
(3) 市售日本納豆商品及其他納豆相關產品分離納
豆激酶生產菌 28
(4) 菌種保存 29
(5) 菌體生長量之測定 29
《1》菌體濃度測定 29
《2》生菌數測定 29
(6) 接種源製備 29
(7) 納豆培養 30
2. 血纖維蛋白平板試驗法 31
3. 納豆激酶活性測定 32
4. 紫外線照射誘導突變法 33
5. 掃瞄式電子顯微鏡 (Scanning Electron Microscopy,
SEM) 觀察 33
(二) 最適生產納豆激酶培養條件之測定 34
1. 固態培養最適條件之測定 34
(1) 最適培養基質之測定 34
(2) 混合培養基質對納豆激酶影響之測定 34
(3) 最適培養溫度、時間之測定 34
(4) 最適接菌量之測定 34
(5) 最適培養相對濕度之測定 35
2. 液態培養最適條件之測定 35
(1) 三角搖瓶最適培養溫度及pH之測定 35
(2) 發酵槽培養試驗 36
(3) 黏度分析 36
(三)、納豆激酶精製及其穩定性之測定 37
1. 納豆激酶製品之精製 37
2. 穩定性之測定 37
3. 納豆激酶製品穩定性之測定 37
(四)、統計分析 38

肆、結果與討論 39
一、高產量納豆激酶生產菌之篩選 39
(一) 高活性納豆激酶生產菌之測定 39
(二) 高活性納豆激酶突變株 40
1. 紫外線照射誘導突變 40
2. 高活性納豆激酶突變株之篩選 41
(三) CM13突變株掃瞄式電子顯微鏡外觀型態之觀察 41
二、CM13突變株最適生產納豆激酶培養條件之探討 51
(一) 固態培養最適條件之測定 51
1. 最適培養基質之測定 51
2. 混合培養基質對納豆激酶活性影響之測定 51
3. 最適培養溫度、時間之測定 52
4. 最適接菌量之測定 52
5. 最適培養相對濕度之測定 52
6.培養過程中納豆激酶與黏度相關性之探討 52
(二) 液態培養最適條件之測定 53
1. 三角搖瓶最適培養溫度及pH值之探討 53
2. 發酵槽培養試驗 53
三、納豆激酶製品之精製及其穩定性之探討 64
(一) 納豆激製品精製之探討 64
(二) 納豆激酶穩定性之探討 64
1. 固態與液態培養之納豆激酶穩定性之探討 64
2. 納豆激酶製品穩定性之探討 66
伍、結論 76
陸、參考文獻 77

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