CN104073444B - Aspergillus oryzae TJTSW001 and the application in Chinese medicine Semen Sojae Preparatum ferments thereof - Google Patents
Aspergillus oryzae TJTSW001 and the application in Chinese medicine Semen Sojae Preparatum ferments thereof Download PDFInfo
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Abstract
Description
技术领域technical field
本发明涉及米曲霉，尤其是涉及一种米曲霉TJTSW001及其在中药淡豆豉发酵中的应用。The invention relates to Aspergillus oryzae, in particular to Aspergillus oryzae TJTSW001 and its application in the fermentation of traditional Chinese medicine Douchi.
背景技术Background technique
淡豆豉是一味常用的传统中药，始载于《名医别录》，历代本草都有记载，它是常用中成药“银翘解毒片”处方的主要药味之一，也是历代《中国药典》的收录品种。本品为豆科植物大豆的成熟种子的发酵加工品，除黑色种皮品系的大豆为主要原料外，还用桑叶、青蒿等药材配以其中进行发酵。中医传统应用本品于临床的功效主治为:解表、除烦、宣发郁热。用于感冒、寒热头痛、烦躁胸闷、虚烦不眠等。Tempeh is a traditional Chinese medicine that is commonly used blindly. It was first recorded in "Famous Doctors", and it has been recorded in successive herbal medicines. It is one of the main medicinal flavors in the prescription of the commonly used Chinese patent medicine "Yinqiao Jiedu Tablets", and it is also included in the "Chinese Pharmacopoeia" of successive dynasties. Variety. This product is a fermented processed product of mature soybean seeds of the leguminous plant. In addition to black seed coat soybeans as the main raw material, mulberry leaves, artemisia annua and other medicinal materials are also used for fermentation. The traditional Chinese medicine application of this product in clinical efficacy and indications are: relieving exterior syndrome, eliminating annoyance, dispelling stagnant heat. For colds, cold and heat headache, irritability and chest tightness, deficiency and insomnia, etc.
大豆异黄酮是大豆的主要生理活性成分，现代研究表明其具有如下功用：Soy isoflavones are the main physiologically active components of soybeans. Modern research shows that they have the following functions:
(1)防癌、肿瘤及抗癌肿瘤作用。(1) Anti-cancer, tumor and anti-cancer effects.
(2)预防骨质疏松。(2) Prevention of osteoporosis.
(3)降胆固醇和心脏保护作用。(3) Cholesterol-lowering and cardioprotective effects.
(4)神经保护及抗神经退行性疾病等等。(4) Neuroprotection and anti-neurodegenerative diseases, etc.
大豆异黄酮在大豆中，主要有两种存在形式，一种为游离型苷元，包括染料木素(又称大豆苷元，金雀异黄素)和黄豆苷，另一种为结合型的糖苷。糖苷主要以丙二酰染料木素，丙二酰大豆苷，染料木苷，大豆苷的形式存在。大豆中主要以结合型的糖苷为主，游离的苷元占比相当少，约占总量的2-3％。需要指出的是，苷元的活性要远高于糖苷。结合型的糖苷需要水解成苷元才能发挥作用。因此，如果能将糖苷水解成苷元的形式，将获得更高的生理活性。郭文勇通过实验发现(中药淡豆豉的质量评价方法及其“解表除烦”作用机制研究,硕士学位论文，2004年)淡豆豉中的异黄酮与其“解表除烦“的功效存在一定联系。因此，获得更高活性的大豆异黄酮苷元，对提高淡豆豉的药效有十分显著的现实意义。Soybean isoflavones in soybeans mainly exist in two forms, one is free aglycon, including genistein (also known as daidzein, genistein) and daidzein, and the other is bound type glycosides. Glycosides mainly exist in the form of malonyl genistein, malonyl daidzin, genistin, and daidzin. Soybean is mainly composed of bound glycosides, and the proportion of free aglycones is quite small, accounting for about 2-3% of the total. It should be pointed out that the activity of aglycones is much higher than that of glycosides. Conjugated glycosides need to be hydrolyzed into aglycones to function. Therefore, if the glycoside can be hydrolyzed into the form of aglycon, higher physiological activity will be obtained. Guo Wenyong found through experiments (Quality Evaluation Method of Traditional Chinese Medicine Light Douchi and Its Mechanism of "Relief Exterior and Elimination", Master's Dissertation, 2004) that there is a certain relationship between the isoflavones in light Douchi and its effect of "relieving appearance and eliminating troubles". Therefore, obtaining soybean isoflavone aglycone with higher activity has very significant practical significance for improving the efficacy of light tempeh.
中药淡豆豉发酵用的微生物，绝大多数均记载为自然发酵的方式，如《中华人民共和国药典》2010版，关于淡豆豉的记载“用煎过的桑叶、青蒿渣覆盖，闷使发酵至黄衣上遍时，取出，除去药渣。洗净，置容器内再闷15-20天，至充分发酵，香气溢出时，取出，略蒸，干燥，即得。”此外，同样的表述在《湖南省中药炮制规范》1983版，《上海市中药炮制规范》2008版中等均出现过。毋庸置疑，上述发酵的过程中存在众多微生物的共同作用。但从长“黄衣”，以及相关研究可以明确，米曲霉在其发酵的过程中占据着十分优势的地位。《浙江省中药炮制规范》2005版则十分明确的表述了接种米曲霉孢子进行发酵。Most of the microorganisms used in the fermentation of traditional Chinese medicine fermented soybeans are recorded as natural fermentation methods. For example, in the 2010 edition of the Pharmacopoeia of the People's Republic of China, the records on fermented soybeans are "covered with fried mulberry leaves and Artemisia annua residues, stuffed to ferment When the yellow clothes are covered, take it out and remove the medicinal residues. Wash it, put it in a container and stuff it for 15-20 days, until it is fully fermented and the aroma overflows, take it out, steam it slightly, and dry it to get it.” In addition, the same expression It has appeared in the 1983 edition of "Hunan Province Traditional Chinese Medicine Processing Standards" and the 2008 edition of "Shanghai Traditional Chinese Medicine Processing Standards". Undoubtedly, there are many microorganisms working together in the above-mentioned fermentation process. However, from the long "yellow coat" and related research, it is clear that Aspergillus oryzae occupies a very dominant position in the fermentation process. The 2005 edition of "Zhejiang Province Traditional Chinese Medicine Processing Standards" clearly expresses the inoculation of Aspergillus oryzae spores for fermentation.
发明内容Contents of the invention
本发明的目的就是为了解决上述问题，提供一种米曲霉TJTSW001，该米曲霉菌株可高产糖苷酶，可以高效的将结合型的大豆异黄酮水解成苷元和糖。The object of the present invention is to solve the above problems and provide a kind of Aspergillus oryzae TJTSW001, the Aspergillus oryzae strain can produce high glycosidase, and can efficiently hydrolyze the conjugated soybean isoflavones into aglycones and sugars.
本发明的另一目的是应用米曲霉菌株TJTSW001发酵生产中药淡豆豉，获得高含苷元的中药淡豆豉。Another object of the present invention is to use the Aspergillus oryzae strain TJTSW001 to ferment and produce the traditional Chinese medicine light fermented soya bean, and obtain the traditional Chinese medicine light fermented soya bean with high aglycone content.
本发明提供的米曲霉菌株是(Aspergillusoryzae)TJTSW001，该菌株已于2014年3月20日保藏于中国微生物菌种保藏管理委员会普通微生物中心，菌种保藏编号为CGMCCNO.8945，保藏地址为北京市朝阳区北辰西路1号院3号中国科学院微生物研究所。The Aspergillus oryzae bacterial strain provided by the present invention is (Aspergillusoryzae) TJTSW001, and this bacterial strain has been deposited in the General Microorganism Center of China Microbiological Culture Collection Management Committee on March 20, 2014. The bacterial strain preservation number is CGMCCNO.8945, and the preservation address is Beijing Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard No. 1, Beichen West Road, Chaoyang District.
该菌株是经过60Co和紫外复合诱变获得。该菌株具有很高的产糖苷酶活性，利用该菌株生产中药淡豆豉可以获得高含苷元的，大幅高于目前文献报道淡豆豉中苷元含量。The strain was obtained through 60Co and ultraviolet compound mutagenesis. The strain has high glycosidase-producing activity, and the traditional Chinese medicine Douchi can be produced by using the strain, which contains aglycone, which is much higher than the content of aglycone in Douchi reported in the current literature.
本发明所提供的米曲霉菌株，是通过60Co和紫外诱变复合诱变获得的。The aspergillus oryzae strain provided by the invention is obtained through compound mutagenesis of 60 Co and ultraviolet mutagenesis.
首先，将生长好的米曲霉孢子用无菌水洗下，制成孢子悬液，然后将该孢子悬液置于辐照剂量为20ky的60Co照射下，进行诱变。诱变结束的孢子悬液，倒入平皿上，在紫外灯的照射下，进行二次诱变。诱变完成后，在黑暗的环境中静止片刻，以防止光复活。Firstly, the grown spores of Aspergillus oryzae were washed with sterile water to make a spore suspension, and then the spore suspension was placed under 60 Co irradiation with an irradiation dose of 20ky for mutagenesis. The spore suspension that has been mutagenized is poured onto a plate, and subjected to secondary mutagenesis under the irradiation of an ultraviolet lamp. After mutagenesis is complete, rest in a dark environment for a short time to prevent photoreactivation.
然后，在筛选培养基上进行筛选培养，筛选培养基以大豆异黄酮为惟一碳源，以(NH4)2SO4为氮源，以在筛选培养基上形成单个菌落为合适稀释度。以菌落大小为挑选依据，选择出目标菌。Then, the screening culture was carried out on the screening medium, which used soybean isoflavone as the sole carbon source and (NH 4 ) 2 SO 4 as the nitrogen source, and the formation of a single colony on the screening medium was the appropriate dilution. The target bacteria were selected based on the colony size.
本发明所获得的米曲霉(Aspergillusoryzae)TJTSW001，可以在以大豆异黄酮为惟一碳源，以(NH4)2SO4为氮源的培养基上生长良好，培养三天，其菌落直径可达3厘米。进一步利用其发酵淡豆豉，测定其糖苷酶。Aspergillus oryzae (Aspergillusoryzae) TJTSW001 obtained by the present invention can grow well on a culture medium with soybean isoflavones as the sole carbon source and (NH 4 ) 2 SO 4 as the nitrogen source. After three days of cultivation, the colony diameter can reach 3 cm. Further use it to ferment light tempeh, and determine its glycosidase.
本发明具有如下有益效果：The present invention has following beneficial effect:
本发明所获得米曲霉(Aspergillusoryzae)TJTSW001，将其运用在中药淡豆豉的生产上，可以获得高含大豆苷元的淡豆豉，从而可以增加其功效性，具有广泛的前景。The Aspergillus oryzae TJTSW001 obtained in the present invention can be used in the production of traditional Chinese medicine Douchi to obtain Douchi high in daidzein, thereby increasing its efficacy and having broad prospects.
具体实施方式detailed description
为了使本发明实现的技术手段、创作特征、达成目的与功效易于明白了解，下面结合具体实施例，进一步阐述本发明。In order to make the technical means, creative features, goals and effects achieved by the present invention easy to understand, the present invention will be further described below in conjunction with specific embodiments.
本发明所提供的米曲霉菌株，是通过60Co和紫外诱变复合诱变获得的。制备过程如下：The aspergillus oryzae strain provided by the invention is obtained through compound mutagenesis of 60 Co and ultraviolet mutagenesis. The preparation process is as follows:
首先，将生长好的米曲霉孢子用无菌水洗下，制成孢子悬液，然后将该孢子悬液置于辐照剂量为20ky的60Co照射下，进行诱变。诱变结束的孢子悬液，倒入平皿上，在紫外灯的照射下，进行二次诱变。诱变完成后，在黑暗的环境中静止片刻，以防止光复活。Firstly, the grown spores of Aspergillus oryzae were washed with sterile water to make a spore suspension, and then the spore suspension was placed under 60 Co irradiation with an irradiation dose of 20ky for mutagenesis. The spore suspension that has been mutagenized is poured onto a plate, and subjected to secondary mutagenesis under the irradiation of an ultraviolet lamp. After mutagenesis is complete, rest in a dark environment for a short time to prevent photoreactivation.
然后，在筛选培养基上进行筛选培养，筛选培养基以大豆异黄酮为惟一碳源，以(NH4)2SO4为氮源，以在筛选培养基上形成单个菌落为合适稀释度。以菌落大小为挑选依据，选择出目标菌。Then, the screening culture was carried out on the screening medium, which used soybean isoflavone as the sole carbon source and (NH 4 ) 2 SO 4 as the nitrogen source, and the formation of a single colony on the screening medium was the appropriate dilution. The target bacteria were selected based on the colony size.
本发明所获得的米曲霉(Aspergillusoryzae)TJTSW001，可以在以大豆异黄酮为惟一碳源，以(NH4)2SO4为氮源的培养基上生长良好，培养三天，其菌落直径可达3厘米。进一步利用其发酵淡豆豉，测定其糖苷酶。Aspergillus oryzae (Aspergillusoryzae) TJTSW001 obtained by the present invention can grow well on a culture medium with soybean isoflavones as the sole carbon source and (NH 4 ) 2 SO 4 as the nitrogen source. After three days of cultivation, the colony diameter can reach 3 cm. Further use it to ferment light tempeh, and determine its glycosidase.
实施例1：Example 1:
米曲霉(Aspergillusoryzae)TJTSW001与其出发菌株在筛选培养基上生长情况比较。The growth of Aspergillus oryzae (Aspergillus oryzae) TJTSW001 and its starting strain on the selection medium was compared.
将米曲霉(Aspergillusoryzae)TJTSW001和其出发菌株同时接种在筛选培养基上(大豆异黄酮为惟一碳源，(NH4)2SO4为惟一氮源)，置于35℃的霉菌培养基上培养，观察其菌落大小(参见表1)。Aspergillus oryzae (Aspergillus oryzae) TJTSW001 and its starting strain were inoculated on the screening medium (soybean isoflavones were the only carbon source, (NH 4 ) 2 SO 4 was the only nitrogen source), and cultured on the mold medium at 35°C , observe the colony size (see Table 1).
表1Table 1
实施例2：Example 2:
米曲霉(Aspergillusoryzae)TJTSW001与出发菌株产糖苷酶活性比较。Comparison of glycosidase activity between Aspergillus oryzae TJTSW001 and the original strain.
取米曲霉TJTSW001和出发菌株，将它们同时接种于斜面试管中，于35℃下，培养5d，待长好后，然后用无菌水洗下表面孢子，将其接种于液态培养基中，液态培养基为6％的黄豆粉。在35℃，180rpm下培养3天。接种于固体培养基中，固体培养组成为玉米芯50，黄豆粉50(重量份)，含水量为50％。在35℃下培养3天，测定其糖苷酶活性(参见表2)。Take Aspergillus oryzae TJTSW001 and the starting strain, and inoculate them into inclined test tubes at the same time, and cultivate them at 35°C for 5 days. The base is 6% soybean flour. Incubate at 35°C, 180 rpm for 3 days. It is inoculated in a solid culture medium, and the solid culture composition is 50 corncobs, 50 parts by weight of soybean powder, and the water content is 50%. After culturing at 35°C for 3 days, the glycosidase activity was determined (see Table 2).
表2Table 2
实施例3：Example 3:
米曲霉(Aspergillusoryzae)TJTSW001与出发菌株生产淡豆豉实验比较。The comparison between Aspergillus oryzae TJTSW001 and the original strains in the production of light tempeh.
取米曲霉TJTSW001和出发菌株，将它们同时接种于斜面试管中，于35℃下，培养5d，待长好后，然后用无菌水洗下表面孢子，将其接种于液态培养基中，液态培养基为6％的黄豆粉。在35℃，180rpm下培养3天。将其作为液态种。Take Aspergillus oryzae TJTSW001 and the starting strain, and inoculate them into inclined test tubes at the same time, and cultivate them at 35°C for 5 days. The base is 6% soybean flour. Incubate at 35°C, 180 rpm for 3 days. Make it a liquid seed.
取桑叶、青蒿个100克，加水煎煮，煎煮3次，合并滤液，将黑豆浸泡其中，直至药汁完全吸干为止。然后黑大豆蒸熟，待其冷却到室温后，将上述液态种拌入其中，在其上覆盖4层纱布，以保持水分。待其表面布满“黄衣”后，取出置于密闭容器内，在50℃下，发酵15-20天。取出，蒸30分钟，然后干燥。最后测定成品中大豆苷元的含量。此实验一共进行三个批次。其最后测定结果如下表3所示：Take 100 grams of mulberry leaves and Artemisia annua, add water to decoct, decoct 3 times, combine the filtrate, soak the black beans in it, until the medicine juice is completely absorbed. Then the black soybeans are steamed, and after it is cooled to room temperature, the above-mentioned liquid seeds are mixed into it, and covered with 4 layers of gauze to keep moisture. After its surface is covered with "yellow clothes", it is taken out and placed in an airtight container, and fermented at 50°C for 15-20 days. Take out, steam for 30 minutes, then dry. Finally, the content of daidzein in the finished product was determined. A total of three batches were carried out in this experiment. Its final measurement result is as shown in table 3 below:
表3table 3
实施例4：Example 4:
米曲霉(Aspergillusoryzae)TJTSW001传代稳定性试验。Passage stability test of Aspergillus oryzae TJTSW001.
将米曲霉(Aspergillusoryzae)TJTSW001传代5次，分别将它们同时接种于斜面试管中，于35℃下，培养5d.待长好后，然后用无菌水洗下表面孢子，将其接种于液态培养基中，液态培养基为6％的黄豆粉。在35℃，180rpm下培养3天。接种于固体培养基中，固体培养组成为玉米芯50，黄豆粉50(重量份)，含水量为50％。在35℃下培养3天，测定其糖苷酶活性(参见表4)。Passage Aspergillus oryzae (Aspergillus oryzae) TJTSW001 for 5 times, inoculate them in inclined test tubes at the same time, and culture them at 35°C for 5 days. , the liquid medium was 6% soybean flour. Incubate at 35°C, 180 rpm for 3 days. It is inoculated in a solid culture medium, and the solid culture composition is 50 corncobs, 50 parts by weight of soybean powder, and the water content is 50%. They were cultured at 35° C. for 3 days, and their glycosidase activity was determined (see Table 4).
表4Table 4
以上所述仅为本发明的优选实施方式，本发明的保护范围并不仅限于上述实施方式，凡是属于本发明原理的技术方案均属于本发明的保护范围。对于本领域的技术人员而言，在不脱离本发明的原理的前提下进行的若干改进，这些改进也应视为本发明的保护范围。The above descriptions are only preferred implementations of the present invention, and the scope of protection of the present invention is not limited to the above-mentioned implementations. All technical solutions belonging to the principle of the present invention belong to the scope of protection of the present invention. For those skilled in the art, some improvements made without departing from the principle of the present invention should also be regarded as the protection scope of the present invention.
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