桦褐孔菌甾类化合物的分析及深层液态发酵条件的优化
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摘要
桦褐孔菌(Pers.:Fr.) Pil. (syn; Fuscoporia oblique (Pers.:Fr.) Aoshima)属于真菌门、担子菌亚门、层菌纲、非褐菌目、多孔菌科、褐卧孔菌属,是广泛存在于欧洲、亚洲和北美洲的一种典型的寄生在落叶树上的白腐菌。在自然界中多以子实体存在,被称为白桦茸。早在十六世纪,俄罗斯和西伯利亚西部民间就有用白桦茸作药物的记载。用该菌的菌核煎出来的药汁可用于治疗癌症和消化系统疾病,并且没有发现毒副作用。目前美国、韩国及日本正加大对该菌的研究,而我国尚处于研究的初始阶段。自美国的Humfeld H等对蘑菇的深层发酵进行了研究,并提出用发酵法来培养蘑菇的菌丝体后,研究者们纷纷用这种方法进行多种菌类的发酵研究。而国内外对桦褐孔菌的深层发酵研究甚少。对于其次级代谢产物甾类化合物及其测定方法更是鲜有报道。
本论文通过对菌株JNPF-IO01进行菌种鉴定,确定其为桦褐孔菌。对其深层发酵培养基进行了优化,并针对其中具有生物活性的六种甾体类化合物建立了相应的分析方法,对其生物合成途径进行了研究,得出如下结论:
(1)以本实验室保藏的菌株JNPF-IO01为出发菌株,经过对其进行形态学和分子生物学的鉴定,确定其与Inonotus obliquus (AY251310)、Inonotus obliquus strain IFO 8681 (AY558593)和Inonotus obliquus strain MDJCBS88 (DQ103883)聚类在一起,位于同一分支,具有很高的同源性,说明菌株JNPF-IO01为桦褐孔菌。
(2)经试验建立了一种采用高效液相色谱(HPLC)分离、测定白桦脂醇、麦角甾醇、胆甾醇、羊毛甾醇、豆甾醇和谷甾醇这六种甾类化合物含量的方法。色谱条件为:以C18柱为固定相,流动相为不同浓度梯度的H2O-CH3OH,流速1.40 mL/min,检测波长202 nm,整个分析是在40 min内完成的。结果表明HPLC法分析甾类化合物的结果具有很好的重复性和回收率。这个方法适合用来快速、准确定量测定桦褐孔菌子实体和发酵菌丝体中的甾类化合物。
(3)正交实验结果表明摇瓶最佳发酵配方为:葡萄糖3.0%,黄豆粉0.25%,蛋白胨0.25%,KH2PO4 0.3%,MgSO4 0.08%,CaSO4 0.08%,初始pH 4.0,接种量为15%(V/V),培养温度为28℃,装液量为100 mL/500 mL。黄豆粉对桦褐孔菌发酵的影响比较大,此时菌丝体的生物量和甾体类化合物的产量分别为11.297 g /L,112.438 mg/L。
(4)通过向桦褐孔菌发酵培养基中加入桦树皮和桦木的不同提取液,能够促进其菌丝体和甾体类化合物的生成,解释了桦褐孔菌子实体初生于树皮下。通过对六种甾体类化合物合成过程中含量的比较,可以看出甾体类化合物生物合成的三条支路之间没有相互抑制作用,在添加提取液后,均出现增长现象。
Inonotus obliquus (Pers.:Fr.) Pil. (syn; Fuscoporia oblique (Pers.:Fr.) Aoshima) is a white rot fungus that belongs to the family Hymenochaetaceae of Basidiomycetes and is a typical plant pathogenic fungus widely spreaded over Europe, Asia and North America. This fungus is usually found as a sterile conk (sclerotia) called‘Chaga’on Betula species in nature. Since the sixteenth century,‘Chaga’has been used as a folk remedy in Russia and western Siberia. A decoction of fungus sclerotia without toxic effects and has been used in treatment of cancers and digestive system diseases. However, this fungus is just investigated in China at present though being explored extensively in American, Korea and Japan. Since American Humfeld H and other workers studied submerged culture of mushroom and put forward to culture mycelia of mushroom by this method, fungi have been cultured in succession. But the researches about I. obliquus are poor in this aspect. Until now it has not been studied the steroids and its quantitative determination method.
In this paper, studies on the strain identification of strain JNPF-IO01 that identified as Inonotus obliquus. To get more mycelia dry weight (MDW) and steroids, the optimum medium for their accumulation was studied. According to six steroids which show various biological activities, appropriate method to rapid determinate the steroids of I. obliquus in submerged culture was established, moreover, steroids of its biosynthetic pathway has been studied. The results were summarized as follows:
(1)The characteristics of morphology and the analysis of DNA sequences suggest that the original strain JNPF-IO01 is identified as Inonotus obliquus.
(2)A high-performance liquid chromatography (HPLC) method was applied to separate these steroids. The procedure was carried out on a reversed-phase C18 column, using a stepwise gradient of water-methanol as mobile phase, with the flow rate of 1.40 ml/min and the detecting wavelength of 202 nm. The analysis was completed within 40 min. The results showed that this method had good reproducibility and satisfactory recoveries for the determination of steroids. This method could be applied to evaluate real samples, and it was rapid, accurate and suitable for the quantitative determination of steroids in the fruiting bodies and submerged-cultured mycelia of Inonotus obliquus.
(3)The optimum medium for the MDW and steroids accumulation was made up of 3.0% of glucoses, 0.25% of soybean flour, 0.25% of peptones, KH2PO4 0.3%, MgSO4 0.08%, CaSO4 0.08%, initial pH was 4.0, inoculation size 15%(V/V), temperature 28℃and medium load amount 100 mL/500 mL shaking flask. Soybean flour has great influence on the fermentation of Inonotus obliquus and the yield of MDW and steroids were repectively 11.297 g /L, 112.438 mg/L.
(4) Steroids were studyed by adding to extraction of birch bark and birch woods in the synthesized routes. The experiment result shows that birch bark contains nutrients which are necessary for growth of Inonotus obliquus. Therefore the extraction of birch bark can improve the growth of MDW and steroids and this can explain fruiting bodies of Inonotus obliquus growing in bark initially. Compared to six steroids, they has shown growth phenomenon after adding extraction of birch bark and birch woods.
本论文通过对菌株JNPF-IO01进行菌种鉴定,确定其为桦褐孔菌。对其深层发酵培养基进行了优化,并针对其中具有生物活性的六种甾体类化合物建立了相应的分析方法,对其生物合成途径进行了研究,得出如下结论:
(1)以本实验室保藏的菌株JNPF-IO01为出发菌株,经过对其进行形态学和分子生物学的鉴定,确定其与Inonotus obliquus (AY251310)、Inonotus obliquus strain IFO 8681 (AY558593)和Inonotus obliquus strain MDJCBS88 (DQ103883)聚类在一起,位于同一分支,具有很高的同源性,说明菌株JNPF-IO01为桦褐孔菌。
(2)经试验建立了一种采用高效液相色谱(HPLC)分离、测定白桦脂醇、麦角甾醇、胆甾醇、羊毛甾醇、豆甾醇和谷甾醇这六种甾类化合物含量的方法。色谱条件为:以C18柱为固定相,流动相为不同浓度梯度的H2O-CH3OH,流速1.40 mL/min,检测波长202 nm,整个分析是在40 min内完成的。结果表明HPLC法分析甾类化合物的结果具有很好的重复性和回收率。这个方法适合用来快速、准确定量测定桦褐孔菌子实体和发酵菌丝体中的甾类化合物。
(3)正交实验结果表明摇瓶最佳发酵配方为:葡萄糖3.0%,黄豆粉0.25%,蛋白胨0.25%,KH2PO4 0.3%,MgSO4 0.08%,CaSO4 0.08%,初始pH 4.0,接种量为15%(V/V),培养温度为28℃,装液量为100 mL/500 mL。黄豆粉对桦褐孔菌发酵的影响比较大,此时菌丝体的生物量和甾体类化合物的产量分别为11.297 g /L,112.438 mg/L。
(4)通过向桦褐孔菌发酵培养基中加入桦树皮和桦木的不同提取液,能够促进其菌丝体和甾体类化合物的生成,解释了桦褐孔菌子实体初生于树皮下。通过对六种甾体类化合物合成过程中含量的比较,可以看出甾体类化合物生物合成的三条支路之间没有相互抑制作用,在添加提取液后,均出现增长现象。
Inonotus obliquus (Pers.:Fr.) Pil. (syn; Fuscoporia oblique (Pers.:Fr.) Aoshima) is a white rot fungus that belongs to the family Hymenochaetaceae of Basidiomycetes and is a typical plant pathogenic fungus widely spreaded over Europe, Asia and North America. This fungus is usually found as a sterile conk (sclerotia) called‘Chaga’on Betula species in nature. Since the sixteenth century,‘Chaga’has been used as a folk remedy in Russia and western Siberia. A decoction of fungus sclerotia without toxic effects and has been used in treatment of cancers and digestive system diseases. However, this fungus is just investigated in China at present though being explored extensively in American, Korea and Japan. Since American Humfeld H and other workers studied submerged culture of mushroom and put forward to culture mycelia of mushroom by this method, fungi have been cultured in succession. But the researches about I. obliquus are poor in this aspect. Until now it has not been studied the steroids and its quantitative determination method.
In this paper, studies on the strain identification of strain JNPF-IO01 that identified as Inonotus obliquus. To get more mycelia dry weight (MDW) and steroids, the optimum medium for their accumulation was studied. According to six steroids which show various biological activities, appropriate method to rapid determinate the steroids of I. obliquus in submerged culture was established, moreover, steroids of its biosynthetic pathway has been studied. The results were summarized as follows:
(1)The characteristics of morphology and the analysis of DNA sequences suggest that the original strain JNPF-IO01 is identified as Inonotus obliquus.
(2)A high-performance liquid chromatography (HPLC) method was applied to separate these steroids. The procedure was carried out on a reversed-phase C18 column, using a stepwise gradient of water-methanol as mobile phase, with the flow rate of 1.40 ml/min and the detecting wavelength of 202 nm. The analysis was completed within 40 min. The results showed that this method had good reproducibility and satisfactory recoveries for the determination of steroids. This method could be applied to evaluate real samples, and it was rapid, accurate and suitable for the quantitative determination of steroids in the fruiting bodies and submerged-cultured mycelia of Inonotus obliquus.
(3)The optimum medium for the MDW and steroids accumulation was made up of 3.0% of glucoses, 0.25% of soybean flour, 0.25% of peptones, KH2PO4 0.3%, MgSO4 0.08%, CaSO4 0.08%, initial pH was 4.0, inoculation size 15%(V/V), temperature 28℃and medium load amount 100 mL/500 mL shaking flask. Soybean flour has great influence on the fermentation of Inonotus obliquus and the yield of MDW and steroids were repectively 11.297 g /L, 112.438 mg/L.
(4) Steroids were studyed by adding to extraction of birch bark and birch woods in the synthesized routes. The experiment result shows that birch bark contains nutrients which are necessary for growth of Inonotus obliquus. Therefore the extraction of birch bark can improve the growth of MDW and steroids and this can explain fruiting bodies of Inonotus obliquus growing in bark initially. Compared to six steroids, they has shown growth phenomenon after adding extraction of birch bark and birch woods.
引文
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