臺灣博碩士論文加值系統

本研究選擇兩株紅麴菌株（BCRC 31615、BCRC 31499），分別以在來米（精白米），蓬萊米（糙米、精白米及發芽米）為基質於30℃培養，探討其色素及二次代謝產物Monacolin K之變化。
　　由市售購得之在來米及糙米（蓬萊米）作為原料，另將糙米分為三份，其中一份送至米廠脫去外層麩皮精白，另一份則浸水兩日使其發芽成為發芽米。米首先泡水蒸熟，接菌後以第六日至第十四日為採樣點，乾燥後保存，取1克麴米加入5毫升乙酸乙酯萃取後分析之。
　　在培養過程中觀察其外觀，發現BCRC31615的顏色比BCRC31499的顏色要深。色素分析上分別以OD500nm及OD400nm偵測紅色色素及黃色色素之吸光值，BCRC31615的色素產量較高，尤其是發芽米。蓬萊糙米及發芽米不利BCRC31499之色素的生成。
　　在Monacolin K的生成上與色素的生成有相似的趨勢。利用BCRC31615接種於在來米及三種不同形式蓬萊米（發芽米、糙米、精白米），以蓬萊發芽米培養第12日有最高產量381 mg/kg，在來米在第12日有下降的現象，隨後在第14日達最大值287 mg/kg。BCRC31499則以在來米培養第十日有最高產量34 mg/kg，蓬萊糙米及發芽米則未超過10 mg/kg。BCRC31615之Monacolin K生產量，無論是以何種基質培養，皆高於BCRC 31499。
然而citrinin常伴隨著紅色色素生成，遂決定分析BCRC31615菌株citrinin之生成變化。在來米、去皮白米、糙米三種基質所產生的citrinin大約在80-120ppm之間，然而以發芽米培養所產生的citrinin最大值接近300ppm。以發芽米培養雖可得較高的monacolin K，但同時也有最高的citrinin產生，且其增加的幅度遠超過其他基質，所以發芽米確實存有特殊成分可以提高BCRC31615的二次代謝產物生成。

In this research, two Monascus sps. (BCRC 31615, BCRC 31499) were choosed for culturing on Japanica and Indica type of rices at 30 ℃ to understand the yields change of pigments and secondary metabolic product, Monacolin K.
The brown rice (Japanica type rice) purchased from local market was divided into three parts, the one was polished to remove the bran, the other part was immersing in water for two days to become germinated. The other kind of polished rice (Indica type rice) was also purchased from local market. Samples of the red rice were taken and dried from 6 ~14 th day after inoculated with Monascus sp. Then 1 gram of red rice was extracted with 5 ml ethyl acetate to obtain the pigment, monacolin K, and citrinin.
We found that the color of red rice cultured by BCRC31615 was deeper than that from BCRC31499 during the culturing process. The absorbance values of red and yellow pigments were determined with OD500nm and OD400nm. It was found that BCRC31615 has higher yields in pigments than BCRC31499. The brown rice and geminated rice were unfavorable for the synthesis of pigment by BCRC31499. The geminated rice has the highest yields of pigment by BCRC31615.
The production curves of monacolin K and pigment had similar trend with time for the brown rice and the germinated rice. 381 mg/kg of monacolin K was achieved for the germinated rice cultured by BCRC31615 at the 12th day of cultivation. While that for the polished Indica rice was 287 mg/kg at the 14th day of cultivation. Monacolin K production from BCRC 31499 was only 34 mg/kg at the 10th day of cultivation for the polished Indica rice. The brown rice and germinated rice were not good in the production of monacolin K. Yield of monacolin K was more by BCRC 31615 than that by BCRC 31499 no matter what kinds of rice were used.
Production curve of citrinin has similar trend as that of pigment. Concentrations of citrinin of red rice from BCRC 31615 were in the range of 80~120 ppm for the polished Indica rice and polished Japanica rice and brown rice. The most citrinin (300 ppm) was obtained from the germinated rice which also achieved the most monacolin K from BCRC 31615. It is suggested that some ingradients are in the germinated rice which are beneficial for the production of secondary metabolites.

CHINESE ABSTRACT……….……………….…………………………i
ENGLISH ABSTRACT…….……………...............………...………….iii
TABLE OF CONTENTS….……………......…….…………..………….v
LIST OF FIGURES AND TABLES..........………………..……………viii
CHAPTER
I Introduction.……………………….....................…………….1
1.1Morphology, taxonomy and life cycle……….…………1
1.2History and the traditional applications.………………..6
1.3Active composition….....………..…..…........………...11
1.3.1 Pigments……………………………………………11
1.3.2 Monacolin K…………………………….………….18
1.3.3 Citrinin…………………………………..………….28
1.3.4 GABA…………………………………..…………..34
1.3.5 Other components…………………………………..35
1.4Attentions for operating………………….……………36
II Material and methods…..………………...……..……………38
2.1 De-hulled Japanica type rice (brown rice), germinated rice and Polished Japanica type rice………….....….………..38
2.2 Colony…………………………………………………...40
2.2.1 Colonies for experiment……………………………40
2.2.2 Composition of culture medium……………..……..40
2.2.2.1 Medium for colony preservation……….……40
2.2.2.2 The cultural medium of topic colony..…….…41
2.2.3 The preparation of solid cultural medium…...……..41
2.3 Equipment...…………………………………….………..41
2.3.1 Culture……………………..……………………….41
2.3.2 Extract……………………..……………………….42
2.3.3 Analysis…………………………………………….42
2.3.4 Others………………………………………………42
2.4 Medicines……..………….………….…..……..………..42
2.5 Methods and conditions of solid culture………………...43
2.5.1 The culture of colony………………………………43
2.5.2 Solid culture……………………..…………………43
2.5.3 The process of culture……………………………...44
2.6 Methods of analysis……………………………………...44
2.6.1 Pigment analysis……………………………………45
2.6.2 Analysis of Monacolin K...........................................45
2.6.3 Analysis of Citrinin………………………………...46
III Results and discussion.…..…………………….…….…....….48
3.1 Cultural process…………………………………...………48
3.2 The analysis of pigment………….….……….…………...58
3.3 Monacolin K analysis....………….…………..…….…….63
3.4 Analysis of Citrinin……………….…………………..…..70
3.5 Discussion…………………….…………………………..74
REFERENCES…........…………………….…………………………76
Fig. 1.1 The life history of Monascus………………………………………………….4
Fig. 1.2 The formation of asexual spore of Monascus rubber………………..………5
Fig. 1.3. The pigments were squeezed from lyses cell of Monascus………………13
Fig. 1.4 Major pigment produced by members of the genus Monascus……………...14
Fig. 1.5 Probable pathway of the biosynthesis of red pigments…………………16
Fig. 1.6 Cholesterol biosynthetic pathway…………………………………………...19
Fig. 1.7 A. Structure of monacolin K, B. The transform pathway of monacolin K….21
Fig. 1.8 Structural analogy between HMG-CoA and β-hydroxyacid form of monacolin K and mechanism of inhibition…………... …………………………23
Fig. 1.9 Base structure of statins and statin side chains linked at C8 (R1) and C6 (R2) of the base structure…………………………………………………………25
Fig. 1.10 Cell cycle regulation by inhibitors of HMG-CoA reductase and proteasome……………………………………27
Fig. 1.11 The structure of Citrinin……………………………………………………30
Fig 3.1.1 The change of appearance of Polished Indica type rice between day 6 to day 14 post inoculated by BCRC31615…………………………………………..…..50
Fig. 3.1.2 The change of appearance of Polished Japonica type rice between day 6 to day 14 post inoculated by BCRC31615…………………………………….……51
Fig. 3.1.3 The change of appearance of De-hulled Japonica type rice between day 6 to day 14 post inoculated by BCRC31615………………………………………….52
Fig. 3.1.4 The change of appearance of Germinated Japonica type rice between day 6 to day 14 post inoculated by BCRC31615……………………………………….53
Fig. 3.1.5 The change of appearance of Polished Indica type rice between day 6 to day 14 post inoculated by BCRC31499………..……………………………………..54
Fig. 3.1.6 The change of appearance of Polished Japanica type rice between day 6 to day 14 post inoculated by BCRC31499………...………………………………..55
Fig. 3.1.7 The change of appearance of De-hulled Japonica type rice between day 6 to day 14 post inoculated by BCRC31499………………………………………….56
Fig. 3.1.8 The change of appearance of Germinated Japonica type rice between day 6 to day 14 post inoculated by BCRC31499…………..…………………….……..57
Fig. 3.2.1 The yields change of red pigment between different cultural medium infected by BCRC31615…………………………………………………………59
Fig. 3.2.2 The yields change of yellow pigment between different cultural medium infected by BCRC31615…………………………………………………………60
Fig. 3.2.3 The yields change of red pigment between different cultural medium infected by BCRC31499…………………………………………………………61
Fig. 3.2.4 The yields change of yellow pigment between different cultural medium infected by BCRC31499……………………………………………………….62
Fig. 3.3.1 The chromatography chart of standard Monacolin K and red rice analyzed by HPLC…………………………………………………………………….…..64
Fig. 3.3.2 Monacolin K calibration curve…………………………………………..65
Fig. 3.3.3 The yields change of Monacolin K between different cultural medium infected by BCRC31615……………………………………………………….66
Fig. 3.3.4 The yields change of Monacolin K between different cultural medium infected by BCRC31499……………………………………………………….67
Fig. 3.3.5 The comparison of pigment and Monacolin K between different cultural medium infected by BCRC31615………………………………….…………….69
Fig 3.4.1 The chromatography chart of standard citrinin and red rice analyzed by HPLC……………………………………………………………………………71
Fig. 3.4.2 Citrinin calibration curve…………………………………………………72
Fig. 3.4.3 The yields change of Citrinin between different cultural medium infected by BCRC31615…………………………………………………………73
Table 1.1 The statistic table of top 10 reasons for causing death in 2003…7

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