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研究生:尚蘿拉
研究生(外文):Laura Elena Sanchez
論文名稱:秀珍菇(Pleurotusostreatus)生長特性與酵素活性之研究
論文名稱(外文):Growth Characteristics and Enzyme Activity of Pleurotus ostreatus
指導教授:王均琍王均琍引用關係徐志宏徐志宏引用關係
指導教授(外文):Chun-Li WangDouglas J. H. Shyu
學位類別:碩士
校院名稱:國立屏東科技大學
系所名稱:熱帶農業暨國際合作系所
學門:農業科學學門
學類:一般農業學類
論文種類:學術論文
論文出版年:2008
畢業學年度:96
語文別:英文
論文頁數:72
中文關鍵詞:秀珍菇抗氧化力酵素活性
外文關鍵詞:Pleurotus ostreatusantioxidant capacityenzyme activity
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秀珍菇為一種可食用的菇類,並具有豐富的營養價值、生物降解的功能和生物科技的潛力,也是世界三大主要生產的菇類之一,本研究室在探討秀珍菇最適合生長之因子、抗氧化能力以及酵素之分析。結果顯示最適合菌絲體生長的溫度是28℃,最適合的酸鹼度為pH7.0,較好的碳源為蔗糖,其次依序為果糖與麥芽糖。較佳的氮源為酵母抽出物,依序為黃豆粉、麥芽抽出物和蛋白腖。在菌菇的生產期間,其生物轉換率約為52%。秀珍菇子實體在80℃烘箱乾燥下其粉末的甲醇萃取液在7.5mg/ml的濃度下,對DPPH表現出90%的抗氧化能力,冷凍乾燥處理的秀珍菇子實體其粉末的甲醇萃取液在10mg/ml的濃度下,表現出對亞鐵離子最佳的螯合能力(90.81%)。在還原力測定的部分,秀珍菇子實體以80℃烘箱乾燥下其粉末的甲醇萃取液濃度在10mg/ml表現出最佳的抗氧化能力。在共軛雙烯法的測定中,最佳的處理為秀珍菇40℃的乾燥處理後,在濃度7.5mg/ml的甲醇萃取液,有80%的抗氧化能力。酵素表現方面,菌絲體在平板培養時會分泌漆酶、纖維素分解酶以及木聚糖酶。在液態培養時,其蛋白質分泌量從第3天至第14天均有變化。在第3天的時候以使用木糖為基質(4.76±0.18μg/ml )時表現出最大量的蛋白質,第7天的時候以使用纖維素(4.6±0.2μg/ml )為基質時表現出最大量的蛋白質,第14天時以使用guaiacol(2166±94μg/ml )為基質時表現出最大量的蛋白質活性。在固態發酵培養的部份,秀珍菇以太空包培養4個月時達到最大的總蛋白質分泌量(2250μg/ml),液態培養下秀珍菇酵素的分泌最大值分別為木聚糖酶2.8U、纖維素分解酶7.2U、漆酶100.0U。以蓮霧木和芒果木為混合的木屑為基質,對秀珍菇作酵素分泌的測試,當培養三個半月時測得11.7U的木聚糖酶,培養一個月時纖維素分解酶的活性為4.97U,漆酶在培養一個月時達到最大活性853U。
Pleurotus ostreatus is an edible mushroom valued for its nutritional properties, bioremediation properties and biotechnological potential. The oyster mushroom is one of the three mushrooms industrially produced worldwide. Studies on Pleurotus ostreatus optimal growth factors, antioxidant capacity and enzyme assays were performed. It was found that the best temperature for mycelial growth was 28 °C, the best pH was 7; the better carbon sources were sucrose, fructose and maltose. The better nitrogen sources were yeast, soybean, malt and peptone. During mushroom production the biological conversion was around 50%. The oyster mushroom extract obtained from fruiting bodies dried at 80 °C showed 90% antioxidant capacity at a concentration of 7.5 mg mL-1 on 1,1-diphenyl-2-picrylhydrazyl radicals. Freeze dried Pleurotus ostreatus fruiting bodies showed the highest chelating effect upon ferrous ions (90.81%) at a concentration of 10 mg mL-1. For the reducing power, the best antioxidant treatment (48%) was from the mushrooms dried at 80 °C at a concentration of 10 mg mL-1. For the conjugated diene method, the best treatment was at 40 °C with an antioxidant capacity of 80% at a concentration of 7.5 mg mL-1. Mycelial growth in plate assay proved the presence of the laccase, cellulase and xylanase enzymes. In submerged fermentation, protein concentration varied from day 3 to day 14. On day 3 the highest protein secreted was found in the treatment in which xylan was used as the substrate (4.76 ± 0.18). On day 7 the treatment with higher protein secretion was the one in which cellulose (4.6 ± 0.2) was used as substrate. On day 14, the highest protein secretion was in the treatment with guaiacol as substrate (2166 ± 94). In solid substrate fermentation, the highest amount of total protein secreted was quantified when the mushroom cultivation bags were 4 months old (2250 μg mL-1). The highest specific activities in submerged fermentation of Pleurotus ostreatus were, for xylanase: 2.8 U, for cellulase 7.2 U , and for laccase 100 U. For xylanase, the specific activity in wax apple mango sawdust of Pleurotus ostreatus was of 11.7 U at 3 months and a half. For cellulase, the specific activity was 4.97 U at 1 month, and for laccase, the specific activity was of 853 U at one month as well.
ABSTRACT (CHINESE) I
ABSTRACT (ENGLISH) II
ACKNOWLEDGEMENTS IV
TABLE OF CONTENTS V
LIST OF FIGURES IX
LIST OF TABLES XIII
1. INTRODUCTION 1

2. LITERATURE REVIEW 3

2.1 Pleurotus ostreatus 3

2.2. Major uses of Pleurotus ostreatus 3

3. MATERIALS AND METHODS 13

3.1 Materials 13
3.1.1 Pleurotus ostreatus 13
3.1.2 Sawdust 13
3.1.3 Machines and apparatus 14
3.1.4 Chemicals 14
3.2 Methods 15
3.2.1 Isolation, purification and culture of mycelium 16
3.2.2 Determination of factors which affect the growth of Pleurotus ostreatus mycelia 16
3.2.2.1 Temperature 16
3.2.2.2 pH value 16
3.2.2.3 Carbon sources 17
3.2.2.4 Nitrogen sources 17
3.2.3 Cultivation and production of Pleurotus ostreatus fruiting body 17
3.2.3.1 Preparation of spawn 18
3.2.3.2 Preparation of cultivation bag 18
3.2.3.3 Inoculation of spawn to cultivation bag 19
3.2.3.4 Management of mushroom house 20
3.2.3.5 Harvest of fruiting body 20
3.2.4 Calculation of biological efficiency (B. E.) 21
3.3 Antioxidant activity 21
3.3.1 Sample preparation 22
3.3.2 Measurement of antioxidant capacity from methanolic extracts 24
3.3.3 Scavenging effect on DPPH (1,1-diphenyl-2-picrylhydrazyl radicals) 24
3.3.4 Chelating effects on ferrous ions 24
3.3.5 Reducing power 25
3.3.6 Conjugated diene method 25
3.4 Enzyme activity 26
3.4.1 Cellulase, xylanase and laccase plate assay 26
3.4.2 Determination of xylanase, cellulase and laccase in submerged fermentation 27
3.4.3 Sample extraction 27
3.4.4 Dinitrosalicylic acid method (DNS) 28
3.4.5 Growth in solid state fermentation 28
3.4.6 Protein concentration determination using Bicinchoninic Acid 30
3.4.7 Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) 31
4. RESULTS AND DISCUSSION 32

4.1 Structure of Pleurotus ostreatus fruiting body 32

4.2 Factors affecting the mycelium growth of Pleurotus ostreatus 33
4.2.1 Effect of temperature on mycelia growth 33
4.2.2 Effect of pH on mycelia growth 35
4.2.3 Effect of carbon sources on mycelia growth 36
4.2.4 Effect of nitrogen sources on mycelia growth 39
4.2.5 The production of Pleurotus fruiting body 41
4.2.6 Antioxidant activity 43
4.2.6.1 Scavenging effect on DPPH (1, 1-diphenyl-2-picrylhydrazyl) radicals 43
4.2.6.2 Chelating effects on ferrous ions 44
4.2.6.3 Reducing power 45
4.2.6.4 Conjugated diene method 46
4.2.7 Enzyme activity 48
4.2.7.1 Cellulase, xylanase and laccase plate assay 48
4.2.7.2 Determination of total protein secreted from Pleurotus ostreatus supplied with xylan, cellulose and guaiacol in submerged fermentation using Bicinchoninic Acid (BCA) 51
4.2.7.3 Determination of total protein secreted from Pleurotus ostreatus supplied with xylan, cellulose and guaiacol in wax apple-mango sawdust using Bicinchoninic Acid (BCA) 55
4.2.7.4 Determination of xylanase, cellulase and laccase specific activities in submerged fermentation 56
4.2.7.4 Determination of laccase xylanase, and cellulase in wax apple- mango sawdust 59
4.2.7.5 Sodium dodecyl sulfate polyacrylamide gel electrophoresis
(SDS-PAGE)……………………………………………………..62

LIST OF FIGURES
Figure 1. Fruiting body of Pleurotus ostreatus presents a convex cap at first, then expanding to a broadly convex cap ………………..... 3

Figure 2. High density level and low density level cholesterol………………………………………………………. 5

Figure 3. Arrangement of lignin, cellulose and hemicellulose in bark of tree……………………………………………………………… 7

Figure 4. Several substrate sources for mushroom production: sawdust (a), rice straw (b), sugar bagasse (c), umbrella plant (d) and coffee pulp(e)………………………………………………………....... 8

Figure 5. Cellulose structure and location in plants ……………………….. 9

Figure 6. Complexity of lignin structure…………………………………… 10

Figure 7. Wax apple and mango wood sawdust……………………………. 13

Figure 8. Burdening system for substrate preparation from sawdust………. 18

Figure 9. Sawdust dispenser (a), bag filling (b), bag sealed with plastic....... 19

Figure 10. Mushroom cultivation bags ready to be inoculated with oyster mushroom mycelium…………………………………………… 19

Figure 11. Cultivation bags inoculated with oyster mushroom spawn……... 20

Figure 12. Fruiting bodies of oyster mushroom (P. ostreatus)……………... 21

Figure 14. Reflux extraction machine……………………………………… 23

Figure 15. Filtering of the mushroom powder using a Büchner funnel filter. 23

Figure 16. Pleurotus ostreatus mycelium staining with 0.1% Congo Red… 26

Figure 17. P. ostreatus mycelium samples containing different substrates: 1% xylan, 1% cellulose, 1% guaiacol in PDA………………….. 27

Figure 18. Homogenization of mushroom substrate sample……………….. 29

Figure 19. Filtration and centrifugation of mushroom substrate sample…………………………………………………………... 29
Figure 20. Oyster mushroom mycelial supernatant sample obtained from the mango- wax apple mix substrate after homogenization, filtration and centrifugation…………………………………. 29

Figure 21. Test tube assay procedure………………………………...…….. 30

Figure 22. Extracted samples in sodium acetate buffer and denatured in SDS-PAGE buffer are loaded into the wells………….….…….. 31

Figure 23. The carpophore (fruiting body) of P. ostreatus at maturity……................................................................................. 32

Figure 24. The anatomy of Pleurotus ostreatus fruiting body under the microscope……………………………………………………… 33

Figure 25. Effect of temperature on mycelium growth of Pleurotus ostreatus 6 days after inoculation…….………………………… 34

Figure 26. Effect of different pH on the mycelium growth of Pleurotus ostreatus 6 days after inoculation………………………………. 35

Figure 27. Effect of pH on Pleurotus ostreatus mycelium growth 10 days after inoculation……………………………………………….... 36

Figure 28. Effect of different carbon sources on the mycelium growth of Pleurotus ostreatus 10 days after inoculation………………….. 38

Figure 29. Effect of different carbon sources on the mycelial growth of Pleurotus ostreatus 4 days after inoculation. Cellulose (a), Corn starch (b), fructose (c), lactose (d), maltose (e), molasses (f), sucrose (g), corn starch (h) and soluble starch (i)………….…… 38

Figure 30. Effect of different nitrogen sources on the mycelium growth of Pleurotus ostreatus 10 days after inoculation………………….. 40

Figure 31. Effect of nitrogen sources on the mycelium growth of Pleurotus ostreatus. …………………..……………………………............ 41

Figure 32. New inoculated mushroom cultivation bag filled with mycelium (a), after 22 days inoculation, mycelium growing throughout the bag (b)……………………………………….…………….. 42

Figure 33. Antioxidant activity of Pleurotus ostreatus dried fruiting bodies at 40 °C, 80 °C and freeze dry on DPPH radicals……………… 44

Figure 34. Chelating effect of Pleurotus ostreatus dried fruiting bodies at 40°C, 80 °C and freeze dry on ferrous ions……………......…… 45

Figure 35. Reducing power of Pleurotus ostreatus dried fruiting bodies at 40°C, 80 °C and freeze dry……………………………………... 46

Figure 36. Antioxidant capacity of Pleurotus ostreatus dried fruiting bodies at 40°C, 80 °C and freeze dry determined by the conjugated diene method……………………..………………… 47

Figure 37. Cellulase activity in P. ostreatus mycelium after 9 days. Notice the halo (clear area) around the growing colony of mycelium…. .49

Figure 38. Xylanase activity in Pleurotus ostreatus mycelium after 10 days. Notice the clear area around the growing colony of mycelium….................................................................................. 50

Figure 39. Laccase activity of Pleurotus ostreatus mycelium after 10 days............................................................................................... 51

Figure 40. Absorbances data from Pleurotus ostreatus mycelium growing in media enriched with 1% guaiacol, 1% cellulose and 1% xylan under submerged fermentation for 3 days…….…………. 52

Figure 41. Absorbances data from Pleurotus ostreatus mycelium growing in media enriched with 1% guaiacol, 1% cellulose and 1% xylan under submerged fermentation for one week…………..... 53

Figure 42. Absorbances data from Pleurotus ostreatus mycelium growing in media enriched with 1% guaiacol, 1% cellulose and 1% xylan under submerged fermentation for two weeks …………... 54

Figure 43. Total protein secreted in mango-wax apple sawdust fermentation during the growth cycle of Pleurotus ostreatus fruiting bodies…………………………………………………... 56

Figure 44. Laccase production of Pleurotus ostreatus mycelium grown on submerged fermentation enriched with 1% guaiacol…................ 57

Figure 45. Xylanase optical density in submerged fermentation in Pleurotus ostreatus mycelium………………………………….. 57

Figure 46. Cellulase optical density in submerged fermentation in Pleurotus ostreatus mycelium………………………………….. 58

Figure 47. Laccase production on mango-wax apple sawdust during the growth cycle of Pleurotus ostreatus fruiting bodies……………. 60

Figure 48. Xylanase and cellulase production on mango-wax apple sawdust during the growth cycle of Pleurotus ostreatus fruiting bodies…........................................................................................ 61

Figure 49. SDS-PAGE from precipitate of P. ostreatus colonized cultivation bag (solid state fermentation) undergoing different growth stages……………..…………………………………….. 62

5. CONCLUSIONS 63

REFERENCES 64

BIOSKETCH OF THE AUTHOR 71

APPENDIX 72
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