摘要
樟芝菌,又名牛樟芝,是我国台湾特有的珍稀食用和药用真菌,可产三萜、多糖、甾醇、苯环类化合物等多种活性成分;樟芝培养物具有保肝、抗癌、调节免疫力、抗炎症等药理活性。近年来,许多学者研究发现,樟芝菌丝体产物具有与子实体相近的药理活性功能。同时,樟芝菌丝体产物中鉴定出的Antrodins和Antroquinonol类化合物,在保肝、抗癌方面具有卓越的疗效。但是目前关于樟芝菌丝体产物中特征活性成分系统的鉴定分析较少,使得樟芝菌丝体产物的发酵调控缺乏目标性,产物的质量控制也不明确。大多数的发酵研究都是关于多糖、菌体量等非特征性活性产物,使得樟芝菌丝体产物的品质始终处于较低水平,市场竞争力差。
本课题的目的是通过对樟芝菌丝体产物活性成分较为全面的鉴定解析,从中筛选出樟芝菌丝体产物的指标化合物,对来源不同的樟芝发酵产物HPLC图谱进行了对比分析;根据筛选出的指标化合物,对樟芝固态发酵和液态发酵两种人工培养方法进行优化,研究工艺条件和产物后处理方式;针对液态发酵产物的不足,建立高通量生产樟芝活性产物的耦合发酵方法,为樟芝的进一步推广打下基础。
本课题主要研究结果如下:
(1)利用柱层析、制备HPLC等技术,对樟芝发酵产物中的活性成分进行了鉴定解析。从樟芝产物中纯化出8个化合物,经质谱、核磁等手段分析,这8个化合物分别为:Ergostatrien-3β-o(lAC-1);Antrodin C(AC-2);Antrodin B(AC-3);Antrodin A(AC-4);Antroquinonol ( AC-5 ) ; Antroquinonol B ( AC-6 ) ;4,7-Dimethoxy-5-methyl-1,3-benzodioxole(AC-7);Ubiquinone-3(AC-8)。
(2)对来源不同的樟芝发酵产物HPLC图谱进行了对比分析。通过分析可知,无论是樟芝固态发酵还是液态发酵产物,其活性物质与樟芝子实体还是有一定的差异,但是也有其自身的特点。樟芝液态发酵产物,其主要活性成分为Antrodins类化合物,尤其以Antrodin C含量最高。樟芝固态发酵产物,其主要活性成分为Antrodins类和Antroquinonol类化合物,各组分的含量较为均衡。
(3)对樟芝固态发酵过程中影响Antrodins的因素进行了优化。实验确定的最适培养基(/1 L锥形瓶)为:大米100 g,黄豆粉0.6 g,K2HPO4 0.05 g,MgSO4 0.05 g。培养基初始含水量为52%,接种量为35%(v/m),培养24 d。发酵过程温度采用变温调控,第一阶段培养温度为28℃,培养16 d后将发酵温度调整为25℃继续培养8 d。在最佳培养条件下,樟芝固态发酵产物中Antrodin A-C的含量高达3096.9 mg/kg、6876.7 mg/kg和8095.7 mg/kg。
(4)利用Plackett-Burman design和Box-Behnken响应面分析法,优化了樟芝液态发酵培养基成分对Antrodin C产量的影响,并建立了模型。得到最优液态发酵培养基为:葡萄糖72 g/L、黄豆粉5.91 g/L、MgSO4 0.614 g/L,樟芝液态发酵培养基理论上产Antrodin C的最大值178.59 mg/L。根据上述回归分析结果和响应面实验特点,在实验水平内安排验证实验,发酵7 d后实际产Antrodin C为177.83±0.32 mg/L,该模型能较好地预测实际产Antrodin C情况。
(5)研究了樟芝液态发酵过程中高溶氧对樟芝活性产物Antrodin C合成的影响。结果显示,三种方式均可以在发酵中后期大幅度提高发酵液溶氧水平,从而提高Antrodin C产量。其中添加氧载体十二烷、流加过氧化氢是较适合樟芝液态发酵的方式。
(6)考察调节细胞通透性对樟芝产Antrodin C的影响。添加0.3%M,胞外Antrodin C含量从41.72%提高到74.60%;摇瓶实验中,在发酵第4 d添加0.5%M,Antrodin C产量高达322.17 mg/L,比对照提高了84.03%。
(7)将调解细胞通透性与原位萃取相结合,提出了能有效提高樟芝液态发酵生产Antrodins和Antroquinonol类化合物的原位萃取发酵技术。优化的耦合发酵体系为:在发酵第4 d添加0.7%表面活性剂M,之后在第5 d加入10%原位萃取剂十二烷,同时在发酵第4 d开始流加0.15 mmol/L/h过氧化氢提高发酵液溶氧。通过耦合发酵,Antrodin C总含量高达455.78 mg/L,而十二烷萃取相中Antrodin C和Antroquinonol的含量分别高达2624.42 mg/L、489.09 mg/L,
(8)对樟芝菌丝体产物的一般性成分进行了分析,实验结果表明,樟芝产物含有丰富的高级脂肪酸、氨基酸、腺苷、活性多糖等营养功能成分,并且在发酵过程中能产生丰富的挥发性香气成分。
Antrodia camphorata, also named Niuchangchih, is an edible and medicinal fungus that only grown on the inner cavity of the Cinnamomum kanehirae Hayata (Lauraceae) in Taiwan. The bioactive compounds identified from A. camphorata including tritrpenoids, polysaccharides, steroids, benzenoids and other metabolites, have exhibit hepatoprotective, anti-cancer, immuno-modulatory and anti-inflammatory activities. Recently, both the mycelia and fruiting bodies of A. camphorata were reported to have similar pharmacological activities. Moreover, the maleic/succinic acid derivatives (Antrodins) and ubiquinone derivatives (Antroquinonols) isolated from the mycelia of A. camphorata have shown excellent hepatoprotective and anti-cancer activities. However, most of studies on the artificial culture of A. camphorata aimed at the non-characteristic compounds such as polysaccharides or biomass, resulting in the relativlely low value and weak mark competitiveness of the mycelial products.
The aim of this work was to isolate and identify the characteristic compounds from the mycelia of A. camphorata based on HPLC analysise. In order to improve the production of these characteristic compounds, the culture conditions of solid state fermentation (SSF) and submerged fermentation (SmF) were optimized, and methods for product treatment were also developed. Furthermore, a coupling fermentation system was established to overcome the deficiency in submerged fermentation system, in which the characteristic compounds were less than that of solid state fermentation system.
The main results of this study are as follows:
(1) By the application of column chromatography and preparative HPLC, eight compounds were isolation and purified. The structures of these compounds were elucidated according to the data of MS and NMR and identified to be: Ergostatrien-3β-ol(AC-1); Antrodin C(AC-2 ) ; Antrodin B(AC-3); Antrodin A(AC-4); Antroquinonol(AC-5) ;Antroquinonol B(AC-6) ; 4,7-Dimethoxy-5-methyl-1,3-benzodioxole(AC-7); Ubiquinone-3(AC-8).
(2) The HPLC of A. camphorata were established based on the comparative analysis of characteristic compounds produced from A. camphorata by different culture methods. The result shows that there some differences in active compounds between mycelium and fruiting body and mycelial product has its own characteristics. The main bioactive components of A. camphorata in submerged fermentation were Antrodins, especially for Antrodin C, while in solid state fermentation were Antrodins and Antroquinonols.
(3) The optimization on culture condition in solid state fermentation was carried out to improve the production of antrodins by A. camphorata. The composition of the medium optimal for Antrodins production were rice 100 g/L, soy bean meal 0.6 g/L, Na2HPO4 0.05 g/L, MgSO4 0.05 g/L with initial moisture content of 52%, inoculum density 35% (v/m) and cultivation for 24 d. The fermentation efficiency was improved by adapting a two-step temperature control strategy, by which the temperature was controlled at 28℃for 16 d and then changed to 25℃for 8 d. Under the optimal conditions, the concentrations of Antrodin A, B and C reached 3096.9 mg/kg, 6876.7 mg/kg and 8095.7 mg/kg, respectively. This is the first report on the Antrodins production by SSF of A. camphorata.
(4) The medium for Antrodin C production by SmF of A. camphorata was optimized using the Plackett-Burman design and Response Surface Methodology.A model was constructed and furthermore, the accuracy and reliability of the model were verified by experiments, in which the results were in good agreement with the predicted ones. By solving the quadratic regression model equation, the optimal conditions were determined as: glucose 72 g/L, soybean powder 5.91 g/L, MgSO4 0.614 g/L and the predicted production of Antrodin C was 178.59 mg/L. After culturing under the optimized condition for 7 d, the prodeuction of Antrodin C reached 177.83±0.32 mg/L, increased by 85.8% compared with that of the former 95.72 mg/L.
(5) Three approaches for improving dissolved oxygen (DO) concentration, including addition of certain proportion of oxygen, oxygen carrier and H2O2, were applied to study the effect of DO on Antrodin C production in SmF of A. camphorate. The results showed that all the three methods could increase the production of Antrodin C by improving DO, in which the addition of oxygen carrier and H2O2 seems more promising in large scale production.
(6) Regulation of cellular permeability exhibited to be another way for improving Antrodin C biosynthesis in SmF of A. camphorate. Results showed that the extracellular Antrodin C content was improved from 41.72% to 74.6% by adding 0.3% surfactant M. Yield of Antrodin C reaeched 322.17 mg/L by adding 0.5% surfactant M in 4 d, according to 35.3% increase than that of control,. In a 5 L fermentor, the yield of Antrodin C and period of biosynthesis were also increased because surfactant M could improve cellular permeability and decrease negative feedback inhibition of high intracellular Antrodin C concentration.
(7) The regulation of cellular permeability and in-situ extration were combined to construct a coupling fermentation system for A. camphorata to produce bioactive metabolites including Antrodins and Antroquinonols, which were similar to the production of SSF. The coupling fermentation system can carry out metabolites synthesis and extraction simultaneously. Hence, the yield of Antrodin C and other compounds were significantly increased. After extraction by ethanol, the extract phase can be recovered and reused in the coupling fermentation system. The optimal conditions of coupling fermentation system were determined as follows: adding 0.7% surfactant M and 10% extractant n-dodecane at day 4 and day 5, respectively, and feeding 0.15 mmol/L/h H2O2 to increase DO. Under the optimal conditions, yield of Antrodin C increased to 455.78 mg/L. Meanwhile, the concentration of Antrodin C and Antroquinonol were 2624.42 mg/L and 489.09 mg/L in extractant n-dodecane respectively.
(8) Systematic studies were carried out on the basic chemical componentseof A. camphorata. The results showed that mycelium of A. camphorata almost contain all types of amino acid and also many kinds of fatty acids, adenosine and polysaccharides. Changes in chemical components of volatile compounds of A. camphorata during solid state fermentation were studied by GC-MS.
引文
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