摘要
本文系统地研究了猪苓、伴生菌和蜜环菌的固体培养特性和液体培养条件，以及影响猪苓菌核和蜜环菌子实体发生的因素，并对影响猪苓酮类成分、多糖和黑色素产生的因素进行了研究。
猪苓菌的营养需求比较简单，在只有碳源和氮源的简单培养基上就能够生长。麦芽汁培养基、GPY培养基和GPC培养基比较适合于猪苓菌固体培养。除了尿素，猪苓对碳源和有机氮源的适应性很强，但在不同的碳源条件下，其菌丝生长速度和气生菌丝形态会有较大差别。玉米浆、酵母膏、奶粉、硫胺素、伴生菌提取物、蜜环菌提取物、活性白土、硅藻土、高岭土都对猪苓固体培养的生长显示出不同程度的促进作用。猪苓的适宜生长温度是25℃。
麦芽汁培养基、GPC培养基和GPY培养基是适合于伴生菌生长的3种培养基。伴生菌对碳源和有机氮源的适应性都很强。伴生菌适宜的生长温度是25℃。伴生菌在胡萝卜培养基上则生长良好，而猪苓菌在胡萝卜培养基上不生长。
用单因子试验和正交试验进行液体培养条件优化，适合猪苓液体培养的培养基配方为：可溶性淀粉20g·L~(-1)，豆饼粉提取液300ml·L~(-1)，玉米浆20ml·L~(-1)，硫胺素0.5g·L~(-1)，pH6.5。适合伴生菌液体培养的培养基配方为：可溶性淀粉20g·L~(-1)，豆饼粉提取液200ml·L~(-1)，玉米浆20ml·L~(-1)，硫酸铵1g·L~(-1)，MgSO_41g·L~(-1)，CaCl_22g·L~(-1)，pH6.0。
进行了3m~3发酵罐的猪苓菌丝体液体培养的中试生产实验。适合作为三角瓶种子制作的液体培养基为玉米面液体培养基(用于摇床培养)或麦芽汁液体培养基(静止培养)。在0.5m~3的一级种子罐中投料0.3m~3，接种量1％，温度25℃，通气量1：0.5，培养至190hr(菌丝干重达到0.23％)适合转接二级罐。二级3m~3发酵罐投料体积为2m~3，接种量15％，温度25℃，通气量1：0.5，间歇搅拌，搅拌转速120rpm，培养115h放罐，菌丝干重达到1％以上。适合作为判断放罐标准的指标是菌丝体的染色形态和发酵液中的氨基氮含量，菌丝干重、发酵液糖含量以及pH可以作为辅助判断指标。猪苓菌丝液体培养时菌丝结成致密的小球状。菌丝滤饼呈乳白色，新鲜的发酵液和菌丝体都有浓郁的苦杏仁味。和真空干燥相比，气流干燥方式更适合于猪苓菌丝体的干燥。
猪苓菌液体摇瓶培养的生长周期为：延迟期为第0～8天，对数生长期为第9～13天，稳定期为第14～18天，第18天之后开始衰亡期。三个生长期的菌丝体的甲醇提取物的HPLC-DAD图谱有差异，其中的检测到的猪苓酮类成分的种类和数量也有较大差异，对数期菌丝体的猪苓酮类成分种类最少，检测到3个成分，稳定期菌丝体和衰亡期菌丝体中都能检测到6个成分。稳定期菌丝体多糖含量最高，比对数期和衰亡期菌丝体高出55％以上。用纸层析法初步测定猪苓菌丝体和发酵液多糖的单糖组成是葡萄糖，与菌核提取的多糖单糖
The solid cultural character and submerged cultural conditions to Grifola umbellata, companion fungus andArmillaria mellea, factors influencing formation in sclerotia of G umbellata and formation of fruitbody in A mellea, and factors influencing formation of polyporusterones, polysaccharides in G umbeUata and melanin in A. mellea were systematically studied in this thesis.
The nutritional requirement is simple for the growth of G umbellata which can grow in media only have carbon source and nitrogen source. Malt-extract agar(MEA), GPC agar and GPY agar were suitable for solid culture to G umbellata. Better adaptability to carbon sources and organic nitrogen source except for urea were observed during the culture of G umbellata, the results showed that the aerial hypha morphous and mycelial growth velocity were different when this funus grew in media with different carbon sources. As growth factors, corn steep liquor, yeast extract, milk powder, thiamin, extract of companion fungi and A mellea mycelia, activated clay, diatomite and kaolin could promote growth of G umbellata. Optimal growth temperature for G umbellata was 25℃.
Three suitable media for the growth of companion fungus were malt-extract agar, GPC agar and GPY agar. Better adaptability to carbon sources and organic nitrogen source were observed during the culture of companion fungi. Suitable temperature for the growth of G umbellata was 25℃.Companion fungus was grew best in carrot-dextrose agar but which was not for G umbellata.
The optimized medium for obtaining high mycela yield of G umbellata was soluble amylum 20 g·L~(-1), bean cake extract 200ml ·L~(-1), corn steep liquor 20 ml·L~(-1), thiamine 0.25 g·L~(-1), pH 6.5. The optimized medium for obtaining high mycela yield of companion fungus was soluble amylum 20 g·L~(-1),bean cake extract 200 ml ·L~(-1), corn steep liquor 20 ml ·L~(-1), ammonium sulfate 1 g·L~(-1), magnesium sulfate 1 g·L~(-1), calcium chloride 2 g·L~(-1), pH 6.0.
Pilot scale production for G umbellata mycelia in submerged culture was experimented in a 3m~3 stirred-tank bioreactor. Inoculums was prepared in the corn meal liquid medium in shake flasks or malt-extract liquid medium in stationary culture flasks. The size of inoculums was about 1% of the first class seeding bioreactor working volume (300L). Seeding culture was carried out at 25 ℃ and aeration rate of 1 : 0.5 for 190h, which mycelial dry weight achieved 0.23%. Working volume of
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