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題 名 | 建立安全有效神麴發酵製程最佳化的研究=The Optimal Fermentation Process for Developing Massa Medicata Fermentata |
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作 者 | 鄧正賢; | 書刊名 | 中醫藥年報 |
卷 期 | 光碟版1:10 2012.10[民101.10] |
頁 次 | 頁200-266 |
專 輯 | 中醫藥產業應用開發 |
分類號 | 414.33 |
關鍵詞 | 神麴; 蛋白酶; 澱粉酶; 脂解酵素; 黃麴毒素; Massa Medicata Fermentata; Shenqu; Aflatoxins; Lipase; Protease; Amylase; |
語 文 | 中文(Chinese) |
中文摘要 | 神麴(Massa Medicata Fermentata)又名六麴、六神麴,由麵粉或麩皮、赤小豆、苦杏仁、鮮青蒿、鮮蒼耳、鮮辣蓼按一定比例混勻後經覆蓋發酵而成的麴劑,含有酵母菌、消化酶、維生素 B 群、麥角固醇 (ergosterol)、揮發油、配糖體等,具有消食健胃、和中止瀉的功效。 本研究團隊在CCMP 94-RD-005 (赭麴毒素A污染中藥材)的計畫中,發現神麴在臺灣收集的25個樣品中檢出21個樣品,赭麴毒素 A濃度介於0.15-45.2 μg/kg,而對神麴高比例的污染狀況,為避免是臺灣單一進口商造成的誤差,在 CCMP95-RD-019的計畫中,採樣擴大到整個大陸地區各省份共 33個樣品,結果顯示,大陸33個神麴樣品中檢出31個含赭麴毒素A,由此可證實神麴為廣泛性的受黴菌毒素污染,而周鳳英 CCMP94-RD-010 (加馬輻射照射對中藥材滅菌及成份影響評估)的計畫中,結果亦顯示神麴藥材其單位重量之最高微生物含量可達107 CFU/g,故本計畫大膽假設神麴傳統發酵炮製過程,有相關毒性菌株參予其中而極易產生黴菌毒素,必須加以釐清改善。 故本計畫之目的在於建立中藥材神麴規格化菌種發酵製程,並以蛋白酶 (Protease)、澱粉酶 (Amylase)與脂解酵素 (lipase)為活性指標,以黃麴毒素 (AflatoxinB1 B2 G1 G2)污染為負指標,篩選優良發酵菌組,建立安全有效的神麴發酵製程最佳化研究,利用現代化製藥科技,提高神麴藥品安全及效能。 本計畫為期一年,以不同領域研究團隊的方式,分三大部分進行進行。 子計畫ㄧ:神麴發酵菌種分離及純化 : 著重於建立神麴菌種分離及純化,並進行菌數檢測,包括總生菌數、總黴菌酵母菌數。在總生菌數(Total Viable Aerobic Count)與總黴菌酵母菌數(Total Yeasts and Molds Count)方面, 26 件樣品中,總生菌數在 1.7×102-9.0×107之間, 11件超過衛生署限量標準(總生菌數<×107 ),不合格率為42.3%,以江西省樣品編號10生菌數狀況嚴重(9.0×107)。26件樣品中,總黴菌酵母菌數在<1×101-1.1×107 之間, 8件超過衛生署限量標準 (總黴菌酵母菌數<×104 ),不合格率為 30.7%,7件樣品總黴菌酵母菌數在<1×101 (未驗出相關黴菌酵母菌數)。 經與子計畫二串連負指標(黃麴毒素污染)及正指標(蛋白質酶 Protease、澱粉酶Amylase、脂解酵素 lipase)活性評估,選定大陸的中藥飲片有限公司所生產的神麴(代號SQTCM)為菌種分離與純化對象。純化共獲的三個菌株, 1株絲狀真菌及2株桿狀細菌供後續研究使用。 1. 代號SQTCM樣品置於Potato Dextrose Agar上,經 30℃, 48小時培養後,分離與純化培養獲得 1種形態之絲狀真菌菌落,2011CT251-1:白色菌落。 2. 代號SQTCM細菌之分離及純化,樣品置於 Trypticase Soy Agar上,經30℃,48小時培養後,分離與純化培養獲得 2株細菌。(1) 2011CT249-1:黃色菌落、表面具光澤;桿狀菌體。(2) 2011CT249-2:米白色菌落、表面不具光澤;桿球狀菌體。並將此樣品提供給子計畫三進行基因序列分析。 子計畫二:神麴傳統發酵菌種篩選及發酵的技術評估:以蛋白質酶(Protease)、澱粉酶 (Amylase)、脂解酵素 (lipase)活性測定為指標,並以黃麴毒素污染為負指標,篩選獲得優良發酵菌組,並比較指標成份辣蓼 (Quercetin)、蒼耳總酚酸(Phenolic acids)、青蒿素(artemisinin) 差異。 結果顯示,本計畫在 100年2-3月間共收集大陸各省份神麴共22件、日本 1件、臺灣3件,共26件樣品。在神麴負指摽-黃麴毒素B1、B2、G1及G2檢測方面,以免疫層析管純化樣品及液相層析儀(附螢光檢出器)檢測神麴負指標成分AF B1、 B2、G1及G2,黃麴毒素 B1在濃度1.0-250.0 ng/mL,得線性迴歸方程式 (Y=mX+b)及相關係數(r)為 Y= 0.6761X+0.3181 (r =0.9971);黃麴毒素B2在濃度0.3-75.0 ng/mL,得線性迴歸方程式(Y=mX+b)及相關係數(r)為Y= 0.1955X+0.1389 (r =0.9994)、黃麴毒素 G1在濃度1.0-250.0 ng/mL,得線性迴歸方程式 (Y=mX+b)及相關係數(r)為Y=0.9570X+0.7353 (r =0.9784),黃麴毒素 G2在濃度0.3-75.0 ng/mL,得線性迴歸方程式(Y=mX+b)及相關係數(r)為Y=0.2208X+0.2498 (r =0.9917),顯示良好線性關係。黃麴毒素 B1、B2、G1、G2同日內及異日間相對標準偏差0.52-3.85 %及1.97-4.17 %,顯示再現性可以接受,神麴藥材添加回收率為73.5-105.6% (Spiked at levels of B1 10 ng/mL、B2 3.0 ng/mL、G1 10.0 ng/mL、G2 3.0 ng/mL),顯示準確性均可以接受。以三倍於指標成分和雜訊之波峰高度比的最小濃度視為儀器的偵測極限,黃麴毒素B1、B2、G1、G2的偵測極限分別為0.1、0.03、0.1、0.03 ng/mL。黃麴毒素標準品 B1、B2、G1、G2液相層析出現順序,依序為 G1 (12.38 min)、G2 (15.02 min)、B1 (18.74 min)、及B2 (20.72 min)。 26件樣品中, 12件受黃麴毒素污染(1.5-76.8 ppb),污染率為 46.1%,5件超過衛生署限量標準15 ppb,不合格率為19.2%,以廣東省樣品編號8污染狀況嚴重,含量為76.8 ppb。 蛋白酶(Protease)、澱粉酶 (Amylase)、脂解酵素 (lipase)活性檢測方面, 26件樣品中,4 件未有任何消化活性,其餘22件樣品澱粉分解酵素活性 Amylase 則在 2.2-89.5 U/g間,蛋白酶(Protease)在0.8-49.8 mU/g間、澱粉酶(Amylase)在2.2-89.5 U/g間、脂解酵素 (lipase)活性7.3-331.2 (mU/g)(表示每克每分鐘的nmole glycerol),以四川省樣品編號21神麴(代號SQTCM)在澱粉酶、脂解酵素表現最好。 子計畫三:神麴優良菌株基因序列分析:基因序列分析結果顯示,經子計畫二負指標 (黃麴毒素污染)及正指標(蛋白質酶 Protease、澱粉酶Amylase、脂解酵素lipase)活性評估,子計畫一SQTCM神麴菌種分離與純化獲得的菌株,細菌菌株經生理生化分析及 16S rDNA序列分析,確定為革蘭氏陽性桿菌芽孢桿菌科(Bacillaceae) 沙芬西菌Bacillus safensis,絲狀真菌為毛黴科Mucoraceae 傘枝黎頭霉真菌Lichtheimia ramosa,並已進行種原保存。 對神麴單一菌種發酵研究中,傘枝黎頭霉真菌Lichtheimia ramose (Absidia corymbifera)菌株以固相發酵槽結果顯示,最佳反應溫度為 40-50 °C,最佳發酵時間為7-10天,傘枝黎頭霉真菌以脂肪分解酵素活性最好,達 285.5±4.9 mU/g,傘枝黎頭霉真菌 Lichtheimia ramose (Absidia corymbifera)為神麴藥材中首次分離出優勢菌株,且無黃麴毒素污染的疑慮,可進一步評估其毒性分析,申請專利並將分離所得之優良菌株提供產業發展之使用。 |
英文摘要 | Massa medicata fermentata (Shenqu) consists of the mixture of fermented powders of wheat flour, Semen Armeniacae Amarum, sprout of Semen Phaseoli, Herba Artmisiae Annuae, Fructus Xanthii and Herba Polygoni Hydropiperis. Shenqu has sweet, pungent and warm properties, and attributive to the spleen and stomach meridians. The actions of Shenqu is to promote digestion and harmonize the stomach. Within the past two projects (CCMP 94-RD-005 and CCMP 95-RD-019), we proceeded the above mycotoxin project to established the method of analysis of Ochratoxin A in Chinese medicines. The method was applied to about 58 samples collected from retail shops in China and Taiwan, Ochratoxin A was detected in 52 samples of Massa Medicata Fermentata, measurable at 0.15-45.2 μg/kg. Evidence has suggested that mycotoxins contaminated in Shenqu produced in traditional processing, related to exposure fungal in fermented preparation. Shenqu was naturally contaminated by fungi that may become toxic for the human organism if the total amount ingested through consumption exceeds a certain tolerable dose. The quality management of Shenqu materials is very important for protectting consumer health. Therefore, a new process for developing Massa Medicata Fermentata was recommended in order to assess the optimal conditions for microbial fermentation of Shenqu. The duration of this research project CCMP-100-RD-021 is approximately one years (20110119-20111231) and divided into three parts. 1. The sub-subject I: To culture and determine the kinds of beneficial microorganism in Shenqu fermentation process. The first part comprises the basic research dealing with identifing and classifing the microorganisms in Shenqu fermentation process. Beside, total plate count test were performed and yeast and mold testing represents the total fungi in sample. The results showed an enormous difference among the different batches of samples in the count of microorganisms.Twenty six of Shenqu were analysed in order to determine the microbial contamination level. The total viable aerobic count of Shenqu varied from 1.7×102-9.0×107 CFU/g and in 11 samples out of 26 was equal to or higher than 107 CFU/g. Rate of unqualified of the total viable aerobic count of Shenqu was 42.3 %. Total yeasts and molds count of Shenqu varied from 1×101-1.1×107 CFU/g and in 8 samples out of 26 was equal to or higher than 104 CFU/g. Rate of unqualified of the total yeasts and molds count of Shenqu was 30.7 %. Microorganisms in the SQTCM Shenqu (No. 21) Sample collected from Sichuan sources were separated and identified. Three strains of microorganisms were isolated. 2. The sub-subject II: To evaluate the effect of isolated unidentified strains and pure inoculation fermentation. The objective of sub-subject II was to evaluate the effect of isolated unidentified strains on promoting digestion. The active ingredients evaluated were lipase activity, protease activity and amylase activity, and screen out the microorganisms for the presence of aflatoxins. In this survey, 26 samples were purchased in Mainland, Taiwan and Japan in order to investigate the aflatoxins contamination and evaluate the enzyme activity in Shenqu. The samples were analyzed of aflatoxins using LC with Post-Column Photochemical Derivatization. The method was applied to 26 samples and aflatoxins were detected in 12 samples, measurable at 1.5-76.8 μg/kg, 5 samples exceed standard requested of the total aflatoxin 15μg/kg. For selecting better fermentation strains, the quality of various Shenqu was evaluated by lipase activity, protease activity and amylase activity. The enzyme activities were found in 24 samples, measurable at 2.2-89.5 U/g (Amylase), 0.8-49.8 mU/g (Protease) and 7.3-331.2 (mU/g) (lipase). With regard to amylase activity and lipase, the SQTCM Shenqu (No. 21) demonstrated higher activity than the others. 3. The sub-subject III: DNA sequencing analysis for the species identification of isolated microorganisms. Identification of Shenqu important microorganisms by ITS sequencing, especially using the ITS region, is reliable and can be used as an accurate alternative to conventional identification methods. Three strains of microorganisms isolated from the SQTCM Shenqu (No. 21), they are Bacillus safensis (Bacillaceae) and Lichtheimia ramosa (Mucoraceae). |
本系統中英文摘要資訊取自各篇刊載內容。