探討光照對Aspergillus ficuum NRRL3135在液態發酵中生產phytase之影響

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研究生：	余程偉 Cheng-wei Yu
論文名稱：	探討光照對Aspergillus ficuum NRRL3135在液態發酵中生產phytase之影響探討光照對Aspergillus ficuum NRRL3135在液態發酵中生產phytase之影響
指導教授：	徐敬衡 Chin-Hang Shu
口試委員:
學位類別：	碩士 Master
系所名稱：	工學院 - 化學工程與材料工程學系 Department of Chemical & Materials Engineering
畢業學年度：	96
語文別：	中文
論文頁數：	83
中文關鍵詞：	Aspergillus ficuum NRRL3135 、phytase
外文關鍵詞：	Aspergillus ficuum NRRL3135, phytase
相關次數：	點閱：10 下載：0
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飼料添加劑phytase酵素加至飼料中，幫助禽畜類吸收金屬離子使用與提升飼料中磷含量的使用率，進而促進禽畜的消化率於產肉率提升。近年來眾多文獻提出同Aspergillus屬之Aspergillus nidulans 、Aspergillus fumigatus、Aspergillus parasiticus、Aspergillus flavus
為含有光接收器之真菌，因此藉由外界不同光源照度，影響菌體生長、產物代謝。
本研究主要目的是在探討光照對於同Aspergillus屬之Aspergillus ficuum NRRL3135生產phytase之影響。先由發酵槽實驗探討白光強度因著光照的距離而調整其照度，研究範圍從0、500、1000、1500、2000、2500 Lux；然而產物phytase活性則隨著照度的增加而隨之提升，達到最適化2000 Lux而後遞減；當操作條件為白光2000 Lux照度時，phytase活性為照度0 Lux時的6倍，比生長速率也提昇約1.93倍之多，然而productivity則提升了7.2倍之多。進而深入探討同光照度2000 Lux之不同光波長，探討範圍由LED Blue light(430 nm-530 nm)、LED Green light(480 nm-590 nm)至LED Red light(590 nm-670 nm)，產物phytase活性則隨著光波長降低而有所提昇，當光波長為LED Blue light(430 nm-530 nm) phytase活性為LED Red light(590 nm-670 nm)時的2.35倍，比生長速率也提昇約1.49倍，而productivity則是提升了2.54倍。綜合以上介紹，由光強度至不同光波長實驗，初步證實光照對於Aspergillus ficuum NRRL3135生產phytase均有所影響。

Adding phytase into feed will help adsorption dietary minerals and increase the P adsorption on poultry,thus improve digestive of poultry.
In recent years, several study indicated fungus of Aspergillus nidulans、Aspergillus fumigatus、Aspergillus parasiticus、Aspergillus flavus are have photoreceptors, so light intensity factor is affecting biomass growth, formation of primary and secondary metabolites.
We are interested in realizing the effects of light intensity on growth biomass and production phytase of Aspergillus ficuum NRRL3135.In this study, we show that the light intensity effects on Aspergillus ficuum NRRL3135 of producing phytase.
In air-lift bioreactor, the white light intensity of lux was controlled by changed the distance of light source, worked in 0、500、1000、1500、2000、2500 lux, phytase activity was increasing with high lux, under optimal conditions,2000 lux, phytase activity was 6 times, specific growth rate was 1.93 times, productivity was 7.2 times at 2000 lux compared with 0 lux.
Studies in different wavelength in 2000 lux,the wavelength range from LED Blue light(430 nm-530 nm)、LED Green light(480 nm-590 nm) 、LED Red light(590 nm-670 nm), phytase activity was increasing with low wavelength,when LED Blue light(430 nm-530 nm), phytase activity was 2.35 times, specific growth rate was 1.49 times, productivity was 2.54 times compared with LED Red light(590 nm-670 nm).

目錄
圖目錄.....................................IX
表目錄....................................XIII
第一章	緒論..................................1
1-1	研究動機……………………………………………………………………1
1-2	研究目的……………………………………………………………………2
第二章	文獻回顧..............................4
2-1真菌..................................................................................................................4
2-1-1真菌應用...............................................................................................4
2-1-2真菌分類表...........................................................................................4
2-1-3 Aspergillus屬........................................................................................5
2-1-4 Aspergillus ficuum NRRL3135.............................................................6
2-2植酸、植酸酶之性質及用途............................................................................9
2-2-1植酸之來源與營養效應.......................................................................9
2-2-2植酸之去除.........................................................................................11
2-2-3植酸酶之作用形式.............................................................................12
2-2-4植酸酶之應用.....................................................................................13
2-3菌體型態(morphology)……………………………………………………..14
2-3-1影響因素及控制球狀菌體形態.........................................................14
2-4 深層液態發酵培養.......................................................................................20
2-5影響發酵的物理化學等環境生長因子........................................................20
2-5-1碳源.....................................................................................................20
2-5-2氮源.....................................................................................................21
2-5-3碳氮比.................................................................................................22
2-5-4溫度.....................................................................................................22
2-5-5 pH值...................................................................................................23
2-5-6通氣量.................................................................................................23
2-5-7光.........................................................................................................24
第三章 實驗規劃、材料與方法………………………27
3-1 實驗規劃.......................................................................................................27
3-1-1 添加陰離子高分子Carbopol搖瓶實驗.......................................... 28
3-1-2 改變培養基體積搖瓶實驗...............................................................28
3-1-3 改變白光照度搖瓶實驗...................................................................29
3-1-4 氣舉式發酵槽白光照度控制實驗...................................................30
3-1-5 氣舉式發酵槽LED 藍、綠、紅光照度控制實驗.............................30
3-2 實驗材料.......................................................................................................31
3-2-1 實驗菌株...........................................................................................31
3-2-2 培養基組成.......................................................................................31
3-2-3 實驗藥品...........................................................................................33
3-2-4 實驗儀器與設備...............................................................................35
3-3 實驗方法.......................................................................................................37
3-3-1 菌種接種及保存方法.......................................................................37
3-3-2 實驗分析方法...................................................................................38
第四章 實驗結果與討論……………………………..43
4-1 添加陰離子高分子Carbopol搖瓶實驗.......................................................42
4-2 改變培養基體積搖瓶實驗...........................................................................46
4-3改變白光照度搖瓶實驗................................................................................48
4-4 氣舉式發酵槽白光照度控制實驗...............................................................49
4-4-1 氣舉式發酵槽實驗之白光強度對Aspergillus ficuum NRRL3135 生
產phytase之影響發酵槽動力曲線圖之比較.................................49
4-4-2 白光強度對發酵時間的影響...........................................................54
4-4-3 白光強度對菌體生長之影響...........................................................54
4-4-4 白光強度對於產物phytase活性的影響..........................................57
4-5 白光波長強度分布.......................................................................................60
4-6 氣舉式發酵槽2000 Lux照度LED 藍、綠、紅光控制實驗........................64
4-6-1氣舉式發酵槽實驗之2000 Lux照度LED  藍、綠、紅光對
Aspergillus ficuum NRRL3135生產phytase之影響發酵槽動力曲線
圖之比較............................................................................................64
4-6-2 光波長對發酵時間的影響...............................................................67
4-6-3 光波長對菌體生長之影響...............................................................67
4-6-4 光波長對於產物phytase活性的影響..............................................69
第五章 結論與建議…………………………………..73
5-1 結論...............................................................................................................73
5-2 建議...............................................................................................................75
第六章 參考文獻……………………………………..76
圖 目 錄
Figure 2-1. 真菌分類表(王，應用微生物學)……………………………5
Figure 2-2. 植酸之分子結構（Bedford, 2000）........................................10
Figure 2-3. 植酸之化學結構及螯合複合物(Thompson,1993)………..10
Figure 2-4. 植酸被植酸酶酵素水解之情形(Liu et al., 1998)………....13
Figure 3-1. 論文實驗架構圖...................................................................27
Figure 3-2. Aspergillus ficuum NRRL3135在agar plate 上之外觀.........31
Figure 3-3. 氣舉式發酵槽裝置實圖.......................................................36
Figure 3-4. 發酵液處理流程...................................................................38
Figure 3-5. 蔗糖濃度標準檢量線……………………………………...40
Figure 3-6. 磷酸二氫鉀濃度標準檢量線……………………………...41
Figure 4-1. 不同Carbopol濃度對Yp/x(U植酸酶活性 g−1菌體重量)之
關係圖...................................................................................45
Figure 4-2. 添加不同Carbopol濃度對菌體形態之影響(A) 0 %，(B)
0.05 %，(C) 0.1 %，(D) 0.2 %，(E) 0.3 % (w/v.)……………45
Figure 4-3. 不同培養基體積對Yx/s(g菌體重量 g−1消耗蔗糖)之關係
圖...........................................................................................47
Figure 4-4. 不同培養基體積對Yp/x(U植酸酶活性 g−1菌體重量)之關
係圖.......................................................................................47
Figure 4-5. 不同白光強度對Yp/x(U植酸酶活性 g−1菌體重量)之關係
圖...........................................................................................48
Figure 4-6. A.ficuum NRRL3135於氣舉式發酵槽白光強度= 0 Lux之
phytase發酵動力曲線圖........................................................50
Figure 4-7. A.ficuum NRRL3135於氣舉式發酵槽白光強度= 500 Lux之
phytase發酵動力曲線圖........................................................50
Figure 4-8. A.ficuum NRRL3135於氣舉式發酵槽白光強度= 1000 Lux
之phytase發酵動力曲線圖....................................................51
Figure 4-9. A.ficuum NRRL3135於氣舉式發酵槽白光強度= 1500 Lux
之phytase發酵動力曲線圖....................................................51
Figure 4-10. A.ficuum NRRL3135於氣舉式發酵槽白光強度= 2000 Lux
之phytase發酵動力曲線圖..................................................52
Figure 4-11. A.ficuum NRRL3135於氣舉式發酵槽白光強度= 2500 Lux
之phytase發酵動力曲線圖..................................................52
Figure 4-12. 不同白光強度對於菌體的比生長速率.............................55
Figure 4-13. 不同白光強度對Yx/s(g菌體重量g−1消耗蔗糖)之關係..56
Figure 4-14. 不同白光強度對Yp/s(U植酸酶活性 g−1消耗蔗糖)之關係
圖.........................................................................................57
Figure 4-15. 不同白光強度對Yp/x(U植酸酶活性 g−1菌體重量)之關
係圖.....................................................................................58
Figure 4-16. 不同白光強度下對於產物phytase產率之關係圖............59
Figure 4-17. 日光燈管於不同照度下光波長與強度分布圖.................60
Figure 4-18. LED 藍光光波長與強度分布圖........................................61
Figure 4-19. LED 綠光光波長與強度分布圖........................................62
Figure 4-20. LED 紅光光波長與強度分布圖........................................62
Figure 4-21. 白光於2000 Lux下LED 藍、綠、紅光光波長與強度分
布圖.....................................................................................63
Figure 4-22. A.ficuum NRRL3135於氣舉式發酵槽LED藍光強度= 2000
Lux之phytase發酵動力曲線圖...........................................63
Figure 4-23. A.ficuum NRRL3135於氣舉式發酵槽LED 綠光強度=
2000 Lux之phytase發酵動力曲線圖..................................65
Figure 4-24. A.ficuum NRRL3135於氣舉式發酵槽LED 紅光強度=
2000 Lux之phytase發酵動力曲線圖..................................65
Figure 4-25. 不同光波長對於菌體的比生長速率.................................68
Figure 4-26. 不同光波長對Yx/s(g菌體重量g−1消耗蔗糖)之關係圖..69
Figure 4-27. 不同光波長對Yp/s(U植酸酶活性 g−1消耗蔗糖)之關係
圖.........................................................................................70
Figure 4-28. 不同光波長對Yp/x(U植酸酶活性 g−1菌體重量)之關係
圖.........................................................................................71
Figure 4-29. 不同光波長下對於產物phytase產率之關係圖................72
表 目 錄
Table 2-1. 不同來源植酸酶之基本特性 (Liu et al., 1998）.....................8
Table 2-2. 飼料原料中植酸磷之含量 (Nelson et al.,1968)…………...11
Table 2-3. 菌絲球形成因子 (楊，2001)………………………………..15
Table 3-1. 黑麴黴Aspergillus ficuum NRRL3135之種瓶培養基組成...32
Table 3-2. 黑麴黴Aspergillus ficuum NRRL3135培養基成份...............32
Table 3-3. 實驗所使用之藥品.................................................................33
Table 4-1. 不同濃度Carbopol對於菌體形態與phytase生成關係表...44
Table 4-2. 不同培養基體積對於菌體生長與phytase生成之關係表...46
Table 4-3. 不同白光照度對於菌體生長與phytase生成之關係表.......48
Table 4-4. 控制不同白光強度下的各發酵參數值.................................53
Table 4-5. 日光燈管於不同照度下光波長與強度分布.........................63
Table 4-6. 控制不同光波長2000 Lux照度下各發酵參數值................66

                                

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