六神曲固态协同发酵工艺优化及物质动态变化研究
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摘要
目的:优化六神曲固态协同发酵工艺并研究发酵过程中物质动态变化。方法:以枯草芽孢杆菌与赛氏曲霉为发酵菌种,蛋白酶和淀粉酶活力为指标,通过单因素实验确定最佳接种比例与发酵时间,采用正交试验优选发酵温度、湿度及接种量。采用HPLC测定六神曲发酵前后苦杏仁苷及青蒿素含量变化,另对发酵过程中两种菌株数量变化与酶活进行检测。结果:最佳发酵工艺为赛氏曲霉和枯草芽孢杆菌接种比例1∶2,时间8 d,温度30℃,湿度85%,接种量10%,蛋白酶与淀粉酶活力分别达到2.16 mg·min~(-1)·g~(-1)和74.84 mg·min~(-1)·g~(-1);苦杏仁苷在发酵后被分解代谢,青蒿素在协同发酵中被代谢但在自然发酵过程中含量基本不变,并且发现两种菌株数量变化与酶活有明显的相关关系。结论:协同发酵工艺条件稳定可行,为六神曲发酵工艺的规范化奠定基础。苦杏仁苷与青蒿素含量均不适合作为质量评价指标。
Objective: To optimize solid-state synergistic fermentation of medicated leaven and investigate the dynamic changes of fermentation substances in the process. Methods: This paper chose the Aspergillus sydowii and Bacillus subtilis for synergistic fermentation. With the activity of protease and amylase as indexes,the single factor experiment was adopted to determine the best vaccination rate and fermentation time. The orthogonal design test was employed to determine the most appropriate fermentation temperature,humidity and inoculation amount. The changes of the contents of laetrile and artemisinin before and after fermentation were detected by HPLC. This paper also detected the changes of the number of the two strains and enzyme activity in fermentation process and found that there was an obvious correlation between them. Result: The best fermentation process: the vaccination rate was 1 ∶ 2( Aspergillus sydowii: Bacillus subtilis). The temperature was 30 ℃. The humidity was 85%. The inoculation was 10% and the fermentation time was 8 days. The protease activity was 2. 16 mg·min~(-1)·g~(-1) and the amylase activity was 74. 84 mg·min~(-1)·g~(-1). Laetrile was decomposed after the fermentation and content of the artemisinin was the same in the process of natural fermentation while that was decomposed in synergistic fermentation. This paper also found that there was an obvious correlation between the number of the two strains and enzyme activity in fermentation process. Conclusion: The synergistic fermentation process was stable and feasible. It will study the solid-state synergistic fermentation of medicated leaven to achieve the goal of standardization of medicated leaven. Both of the contents of laetrile and artemisinin can not be regard as the quality evaluation indexs.
Objective: To optimize solid-state synergistic fermentation of medicated leaven and investigate the dynamic changes of fermentation substances in the process. Methods: This paper chose the Aspergillus sydowii and Bacillus subtilis for synergistic fermentation. With the activity of protease and amylase as indexes,the single factor experiment was adopted to determine the best vaccination rate and fermentation time. The orthogonal design test was employed to determine the most appropriate fermentation temperature,humidity and inoculation amount. The changes of the contents of laetrile and artemisinin before and after fermentation were detected by HPLC. This paper also detected the changes of the number of the two strains and enzyme activity in fermentation process and found that there was an obvious correlation between them. Result: The best fermentation process: the vaccination rate was 1 ∶ 2( Aspergillus sydowii: Bacillus subtilis). The temperature was 30 ℃. The humidity was 85%. The inoculation was 10% and the fermentation time was 8 days. The protease activity was 2. 16 mg·min~(-1)·g~(-1) and the amylase activity was 74. 84 mg·min~(-1)·g~(-1). Laetrile was decomposed after the fermentation and content of the artemisinin was the same in the process of natural fermentation while that was decomposed in synergistic fermentation. This paper also found that there was an obvious correlation between the number of the two strains and enzyme activity in fermentation process. Conclusion: The synergistic fermentation process was stable and feasible. It will study the solid-state synergistic fermentation of medicated leaven to achieve the goal of standardization of medicated leaven. Both of the contents of laetrile and artemisinin can not be regard as the quality evaluation indexs.
引文
[1]芦艳卿,王志红,季兰芳.含六神曲的中成药卫生标准的研究[J].中医药研究,1993(4):1007-6468.
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[3]张南方,韩顺意,张义生.六神曲质量标准研究[J].中医学报,2015,30(1):98-100.
[4]高慧.神曲发酵及炮制工艺研究[D].沈阳:辽宁中医药大学,2003.
[5]王秋红,苏阳,王荔慧,等.六神曲中真菌的分离与鉴定[J].中国实验方剂学杂志,2014,20(7):122-127.
[6]陈娟,焦晓林,杨春勇,等.分离培养方法结合PCR-SSCP技术分析六神曲中的真菌类群[J].中国中药杂志,2014,39(21):4169-4173.
[7]邬吉野,李莹,王德馨,等.六神曲的发酵菌种分离及纯种发酵考察[J].中国实验方剂学杂志,2013,19(16):12-14.
[8]张丽霞,高文远,王海洋.六神曲中酵母菌的鉴定[J].中国中药杂志,2012,37(13):1928-1931.
[9]黄国能.神曲等药曲中消化酶的检测与质量标准的探讨[J].中成药,1981,4(5):18-20.
[10]府炳荣.影响中药“神曲”功效的因素分析[J].抗感染药学,2009,6(4):237-240.
[11]徐云,郑璐,相宏宇,等.六神曲发酵过程中5种消化酶的动态分析[J].中国酿造,2012,31(10):43-45.
[12]王海洋,高文远,张丽霞.六神曲不同的制备工艺对其淀粉酶活力的影响[J].中国中药杂志,2012,37(14):2084-2087.
[13]刘腾飞,高慧,刘晓瑜,等.响应面法优化六神曲发酵工艺[J].中药材,2014,37(10):1757-1761.
[14]程亦雄,张婧,戚岑聪,等.赛氏曲霉深层发酵神曲的工艺优选[J].中国实验方剂学杂志,2013,19(19):42-45.
[15]周德庆.微生物学实验手册[M].上海:上海科学技术出版社,1986:353.
[16]练晶军.六神曲质量特征及发酵变化研究[D].北京:北京中医药大学,2011.
[17]陈缤.神曲的炮制工艺研究[D].沈阳:辽宁中医学院,2005.
[18]刘金磊,李典鹏,韦霄,等.黄花蒿中青蒿素含量的RPHPLC法测定[J].广西植物,2007,27(5):808-810,724.
[19]赵德安.纯种发酵、混合发酵与传统发酵食品[J].中国酿造,2010,29(9):15-17.
[20]张英,侯红萍.微生物混合发酵生产纤维素酶的研究进展[J].酿酒,2010,37(3):20-23.
[21]何华美,李淑珍,陈丹霞,等.芽孢杆菌混合发酵工艺条件的优化[J].中国酿造,2014,33(4):56-60.
[22]贾丹丹,黄春敏,刘英,等.六神曲炮制过程中微生物的分离与鉴定[J].中国现代中药,2016,18(3):357-361.
[2]刘振启,刘杰.六神曲的炮制工艺与混乱品的探讨[J].首都医药,2012,19(5):50-50.
[3]张南方,韩顺意,张义生.六神曲质量标准研究[J].中医学报,2015,30(1):98-100.
[4]高慧.神曲发酵及炮制工艺研究[D].沈阳:辽宁中医药大学,2003.
[5]王秋红,苏阳,王荔慧,等.六神曲中真菌的分离与鉴定[J].中国实验方剂学杂志,2014,20(7):122-127.
[6]陈娟,焦晓林,杨春勇,等.分离培养方法结合PCR-SSCP技术分析六神曲中的真菌类群[J].中国中药杂志,2014,39(21):4169-4173.
[7]邬吉野,李莹,王德馨,等.六神曲的发酵菌种分离及纯种发酵考察[J].中国实验方剂学杂志,2013,19(16):12-14.
[8]张丽霞,高文远,王海洋.六神曲中酵母菌的鉴定[J].中国中药杂志,2012,37(13):1928-1931.
[9]黄国能.神曲等药曲中消化酶的检测与质量标准的探讨[J].中成药,1981,4(5):18-20.
[10]府炳荣.影响中药“神曲”功效的因素分析[J].抗感染药学,2009,6(4):237-240.
[11]徐云,郑璐,相宏宇,等.六神曲发酵过程中5种消化酶的动态分析[J].中国酿造,2012,31(10):43-45.
[12]王海洋,高文远,张丽霞.六神曲不同的制备工艺对其淀粉酶活力的影响[J].中国中药杂志,2012,37(14):2084-2087.
[13]刘腾飞,高慧,刘晓瑜,等.响应面法优化六神曲发酵工艺[J].中药材,2014,37(10):1757-1761.
[14]程亦雄,张婧,戚岑聪,等.赛氏曲霉深层发酵神曲的工艺优选[J].中国实验方剂学杂志,2013,19(19):42-45.
[15]周德庆.微生物学实验手册[M].上海:上海科学技术出版社,1986:353.
[16]练晶军.六神曲质量特征及发酵变化研究[D].北京:北京中医药大学,2011.
[17]陈缤.神曲的炮制工艺研究[D].沈阳:辽宁中医学院,2005.
[18]刘金磊,李典鹏,韦霄,等.黄花蒿中青蒿素含量的RPHPLC法测定[J].广西植物,2007,27(5):808-810,724.
[19]赵德安.纯种发酵、混合发酵与传统发酵食品[J].中国酿造,2010,29(9):15-17.
[20]张英,侯红萍.微生物混合发酵生产纤维素酶的研究进展[J].酿酒,2010,37(3):20-23.
[21]何华美,李淑珍,陈丹霞,等.芽孢杆菌混合发酵工艺条件的优化[J].中国酿造,2014,33(4):56-60.
[22]贾丹丹,黄春敏,刘英,等.六神曲炮制过程中微生物的分离与鉴定[J].中国现代中药,2016,18(3):357-361.