摘要
蛹虫草(Cordyceps militaris)是一种重要的中药,也是名贵中药冬虫夏草(Cordyceps sinensis)的常用替代品。蛹虫草含有虫草多糖、虫草素、核苷类物质、麦角甾醇、虫草酸等多种有效成分,这些有效成分含量是蛹虫草子实体品质的重要指标。蛹虫草的人工培养方式包括液体发酵和固体发酵,有关蛹虫草有效成分含量的影响因素的研究在液体发酵中较多,而固体发酵中大多仅限于对子实体生长条件优化。本文研究了培养条件及培养基对蛹虫草子实体生长及有效成分合成的影响,为高产优质的蛹虫草子实体和主要活性物质生产提供一定的基础依据。
1.研究了培养条件(光照、温度、培养时间)对蛹虫草原基形成、子实体生长及有效成分含量的影响,主要结果如下：
(1)光照(光强、光照时间及光质)是影响蛹虫草子实体生长及有效成分合成的重要因素。光照越强,虫草多糖、腺苷及类胡萝卜素含量越高。较弱的光照则更有利于子实体产量提高和虫草素、麦角甾醇、可溶性蛋白及游离氨基酸合成。延长每天光照时间对原基形成、类胡萝卜素、多糖、腺苷及虫草酸的合成有促进作用,而对子实体生长及可溶性蛋白合成则有抑制作用。对于多糖、虫草素及类胡萝卜素的合成,白炽光显著优于黄光和红光,而对麦角甾醇和游离氨基酸则相反。综合考虑,确定蛹虫草培养适宜的光照条件为：白炽光、光照强度为200 lx,每天光照时间分阶段设置,原基形成阶段为24 h／d,子实体生长阶段16 h／d为最佳。
(2)不同培养温度(20℃、22℃、24℃和26℃)对蛹虫草子实体产量和不同的有效成分含量的影响不同。当温度从20℃升高到26℃时,蛹虫草原基不能正常形成。从20-24℃间,子实体产量和虫草酸含量随着温度的升高而显著下降；对于多糖、虫草素含量,3种培养温度间都有显著性差异,22℃为最佳；20℃与22℃温度条件下培养的子实体腺苷、类胡萝卜素含量显著高于24℃条件下的,可溶性蛋白则相反；对于游离氨基酸的合成,22℃显著优于20℃与24℃。综合分析子实体产量及有效成分含量,确定蛹虫草子实体培养的严格温度区间为20-22℃。
(3)子实体产量及有效成分含量随培养时间的变化而变化。子实体在接种后第40 d达到最大产量,除了多糖外,其他有效成分均能在子实体生长旺盛期大量积累,但随着培养时间延长虫草素含量增加幅度较大。除虫草素、可溶性蛋白及游离氨基酸外,其他5种成分含量达到最高后随着培养时间的延长而下降,培养时间超过40 d后子实体颜色开始变黑。综合子实体产量及颜色、虫草素含量及其他成分,确定最佳采收时间应选择子实体达最大产量后5 d即接种后培养45 d左右。
2.研究了培养基(碳源、氮源、无机盐、维生素、pH)对子实体生长及有效成分含量的影响,主要结果如下：
(1)不同的碳源对子实体生长及其不同有效成分含量的影响不同。对子实体生长,麦芽糖与甘露醇最好。在保证子实体产量的前提下,分析对比麦芽糖与甘露醇对虫草素、腺苷及虫草酸等各种有效成分的合成与积累作用,确定最合适的碳源为甘露醇。
(2)各种氮源添加物对蛹虫草生长及不同有效成分合成的作用不同。对子实体产量、游离麦角甾醇、虫草素和可溶性蛋白的含量,有机氮源的作用要显著优于无机氮源,而对虫草酸的合成则相反。有机氮源中鲜蛹能促进5种有效成分(多糖、虫草素、虫草酸、可溶性蛋白及类胡萝卜素)合成,无机氮源中磷酸氢二铵对4种有效成分(虫草酸、腺苷、游离氨基酸及类胡萝卜素)的积累有明显促进作用。则最佳的有机氮源添加物为鲜蛹,无机氮源为磷酸氢二铵。
(3)维生素作为微量生长因子对蛹虫草生长有明显影响,但对有效成分的合成影响作用不大。相比其他维生素,VB1能促进3种有效成分(虫草酸、游离氨基酸及类胡萝卜素)的合成,对子实体生长也有促进作用,最终选VB1作为最佳添加物。
(4)不同的无机盐对不同的有效成分积累影响不同。添加的所有无机盐中,KH2PO4和NaNO3能促进合成的有效成分种类最多,KH2PO4能促进虫草多糖、虫草素、腺苷、可溶性蛋白及类胡萝卜素的合成,对子实体生长也有促进作用,NaNO3对多糖、腺苷、虫草酸、可溶性蛋白、类胡萝卜素及游离麦角甾醇的合成有明显促进作用,但不利于子实体生长,因此添加KH2PO4最佳。
(5)培养基pH对不同的有效成分合成的影响规律不同。对虫草多糖、虫草素、虫草酸、麦角甾醇、游离氨基酸及类胡萝卜素合成,酸性条件更有利：中性条件下,可溶性蛋白含量最高。综合考虑,蛹虫草培养基pH应调在5.5-6.5之间。
Cordyceps militaris is one of the important traditional Chinese medicine (TCM), also the common substitutes of expensive TCM Cordyceps sinensis. Cordyceps militaris contains polysaccharides, cordycepin, nucleoside substances, ergosterol, cordyceps acid, and other active ingredients, these effective ingredients are important indicator of the quality of Cordyceps militaris fruiting bodies. The artificial cultivation Cordyceps militaris including liquid fermentation and solid-state fermentation, research on the effective constituents of the Cordyceps militaris mainly in liquid fermentation, while the solid state fermentation mostly focus on the optimization growth conditions of the fruitbody. The paper studies on the effect of cultivating conditions and medium to the gorwth of fruitbody Cordyceps militaris and its effective components, providing certain basis of synthesis of high quality Cordyceps militaris and the main active material production base.
Studied the culture conditions (light, temperature, cultivating time) on the primordium formation and fruitbody growth and effective ingredient content, the main results are as follows:
(1) Illumination is one of the most impotant factors of Cordyceps militaris fruitbody growth and effective component synthesising. The stronger light, polysaccharide, adenosine, carotenoids content was higher, the weaker light is more conducive to yield increase and cordycepin, ergosterol, soluble protein and free amino acid synthesis. Extending the time of light will boost the growth of carotenoids, polysaccharide, adenosine and cordyceps acid synthesis, but restrain the fruitbody growth and soluble protein synthesising. For synthesis of polysaccharide, cordycepin and carotenoids, the incandescent light is much better than yellow and red light, superior to ergosterol and free-amino acids oppositely. Comprehensive consideration, determine the appropriate illumination conditions for:the incandescence light of 200 lx, the primordium forming stage for 24 h/d, while 16 h/d for fruitbody growth stage is the best.
(2) Different temperature (20℃、24℃、22℃and 26℃) have different effect to the fruitbody yield and effective ingredients synthesis. When the temperature increases from 20℃to 26℃, the primordium can not be properly formed. From 20-24℃, the fruiting body and cordyceps acid of production decreased significantly as the temperature increasing; for the content of polysaccharides, cordycepin, it has significantly difference between those three culture temperatures, while,22℃for the best; 20℃and 22℃, culture temperature, the bodies of adenosine, carotenoid content are significantly higher than that of 24℃, soluble protein is opposite; for the synthesis of amino acids,22℃and 20℃was significantly higher than 24℃. Comprehensive analysis of fruiting body yield and composition, to determine Cordyceps militaris cultured strict temperature range 20-22℃.
(3) The production and effective ingredient content varied by the cultivation time. The fruitbody after inoculation 40d to maximum yield, besides polysaccharides, other effective ingredients are accumulated greatly at the esse bloom period, but the cordcepin increased lager as prolonger the culrivation time. Except for cordycepin, soluble protein and amino acid, the other 5 elements reached their highest level then turned down as the extension of time, after more than 40d the fruit color went black. Considing the production and color, content of cordycepin and other ingredients content, determined the optimal harvest time should choose the maximum yield of fruitbody after 5 d, that's to say, about 45 days after inoculation cultivation.
2 Researched the effect of substrate (carbon sources, nitrogen source, inorganic salt, vitamin, pH) on the growth of fruitbody and effective ingredient content, the main results are as follows:
(1) For the fruitbody growth, maltose and mannitol is the best. Different carbon source have vary influence on different availability composition synthesis,. Analyse and contrast the effect of maltose and mannitol on cordycepin adenosine cordyceps acid and various effective component synthesis and accumulation, determine the most appropriate carbon source is mannitol.
(2) All sorts of nitrogen additive have different effect to the grow and effective ingredient of Cordyceps militaris synthesis. Considing the fruitbody production, free ergostrerol, cordycepin and soluble protein content, organic nitrogen source will significantly better than inorganic nitrogen source, while the cordycep acid is opposite. Organic nitrogen source of fresh pupa can promote 5 kind of effective components's synthesis (polysaccharide, cordycepin, cordycep acid, soluble protein and carotenoids), inorganic nitrogen source of synthesis and diammonium phosphate have obvious role in promoting the accumulation of 4 kinds of effective ingredients (cordyceps acid, adenosine, free amino acid and carotenoids). So the best source of organic nitrogen additive is fresh pupa, and inorganic nitrogen source is diammonium phosphate.
(3) Vitamins as the trace growth factor for Cordyceps militaris growth has obvious effect. VB1 can promote 3 kinds of the effective components (cordycep acid, free amino acid and carotenoids), also promote the fruitbody growth, finally VB1 chosen as the best additive.
(4) Of different inorganic salts on the cumulative effects of different active ingredients in different. Through contrast KH2PO4 and NaNO3, which can promote most types of effective components, by the effect of polysaccharide, cordycepin, adenosine of effective ingredients of synthesis and fruitbody, finally the former chosen as the better additive.
(5) Medium pH value has various different effects on the synthesis of the active ingredient. For the synthesis of polysaccharide, coedycepin, cordyceps acid, free ergosterol, free amino acid and carotenoids acidic conditions, acidity is more advantageous; On the neutral condition, soluble protein content is the highest. Comprehensive consideration, medium pH Cordyceps militaris should be adjustable betweent 5.5-6.5.
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