【作者】 颜松；

【导师】 胡开辉；

【作者基本信息】 福建农林大学 ， 微生物学， 2008， 硕士

【摘要】 真姬菇（Hypsizigus marmoreus）是一种营养丰富的食用兼药用的珍稀食用菌,也是一种极具潜力的生产品种。然而,由于传统的真姬菇栽培方式——固体菌种栽培具有周期长、效率低、商品价值低下等存在的问题,所以限制了真姬菇的工厂化和规模化发展。本研究通过正交试验等方法对真姬菇液体发酵工艺进行了优化,并与真姬菇传统固体菌种的栽培模式进行了比较,并通过化学、GC-MS化学指纹图谱等分析手段对其发酵产物化学成分进行了分析,这为真姬菇的产业化生产以及进一步开发真姬菇功能性食品提供了理论基础和应用价值。主要研究结果如下:1、研究了营养性因子对真姬菇真9603菌株深层发酵的影响,通过单因素和正交试验的方法对其生长的最适培养基进行了优化。采用深层培养方法筛选出了真9603菌株液体深层发酵较优的碳源和氮源,研究结果表明,马铃薯、葡萄糖、酵母粉有利于真姬菇真9603菌株菌丝体的生长,进一步的正交优化结果确定了真姬菇真9603菌株深层发酵最适的培养基配方为:马铃薯20%、葡萄糖3%、酵母粉0.2%、KH₂PO₄0.1%、MgSO₄·7H₂O0.1%。2、采用摇瓶培养方法对真姬菇真9603菌株的深层培养条件进行了研究,结果表明,真姬菇真9603菌株深层培养的最佳条件是:摇瓶柜的转速为120r/min,培养温度20℃,起始pH值6.0-7.5（一般不需调pH值）,接种量为5mL左右（250mL锥形瓶,100mL瓶装量）,即接种量5%,摇瓶装量为100mL（250mL锥形瓶）。3、研究了真姬菇真9603菌株液体菌种培养过程中菌球数量、pH值、还原糖含量、氨基氮含量以及相关酶活性等指标的动态变化情况。研究结果表明,真姬菇液体发酵产菌球的数量和活性与其它指标具有一定的相关性。当培养液中的还原糖含量和氨基氮含量分别为0.98g/100mL和7.52mg/100mL,羧甲基纤维素酶活性和蛋白酶活性分别为7.85U和12.6U,pH为5.20时,菌球数量（60个/mL）与活性达到最高,发酵终点确定为培养后第7天。4、进行了真姬菇液体菌种与固体菌种的对比应用试验,并进行了相关的效益分析。研究结果表明,与采用固体菌种的栽培模式相比,采用液体菌种栽培具有周期短、出菇整齐、生产效率高等优势,这充分说明了真姬菇液体菌种具有良好的应用前景。接种量以每袋15mL的效果最好;在0-25℃的温度范围内,真9603菌株液体菌种的活力与保藏温度和保藏时间呈负相关,其最适贮藏温度为0-10℃,最适的保藏时间为3-9天;其液体菌种穿刺接种的日均生长2.21mm,液体菌种表面接种的日均生长1.71mm,固体菌种菌丝的日均生长1.21mm。其液体菌种穿刺接种的生物学效率可以达到64.80%,液体菌种表面接种的生物学效率可以达到63.40%,固体菌种菌丝的生物学效率仅为51.00%。真姬菇在液体菌种栽培模式下出菇比固体栽培模式下更整齐,而其它的性状差异并不明显。5、对真姬菇真9603菌株的发酵液化学成分进行初步测定,系统的预试验结果表明发酵液中含有氨基酸、蛋白质及多肽、有机酸、皂甙、黄醌及其甙类物质、内酯、香豆素及其甙类,蒽醌及其甙类、挥发油、酚类物质、强心甙、植物甾醇,三萜等物质;不含生物碱、鞣质、氰酐等物质。通过GC-MS测试,确定真姬菇真9603菌株发酵液的挥发性气体中含有烷烃类、醇类、酮类、醛类及其它类别的一些物质,其中醇类物质和酮类物质的含量相对较高,分别为7.46%和7.13%。本实验也对真姬菇真9603菌株发酵液的氯仿萃取相中的物质进行了成分鉴定,结果表明,氯仿萃取相中含有烷烃类物质,酚类物质,酯类物质,醇类物质、酮类物质和其它类别的一些物质,其中烷烃类物质和酚类物质占多数,分别为12.76%和10.63%,其中很多物质已经被证明具有良好的抗氧化、抗衰老等保健功能,这为进一步开发真姬菇功能性食品提供了一定的理论基础和应用价值。更多还原

【Abstract】 Hypsizigus marmoreus is one of the nutritious rare fungus for edible and officinal use. It’s a kind of potential producing breed. However, the traditional planting method of Hypsizigus marmoreus, that is solid spawn planting method, is of long cycle, low efficiency, and low commercial value, therefore it restricts the industrialization and scale of Hypsizigus marmoreus. So the research on technics and application of Hypsizigus marmoreus liquid spawn was carried through. We used the orthogonal test to optimize the liquid fermentation technics of Hypsizigus marmoreus. Then we compared it to the traditional solid spawn planting method of Hypsizigus marmoreus. And we used the chemistry and GC-MS chemical fingerprint collection of illustrative plates method to analyse the chemical components of the ferment production. This research afforded the theory foundation and application value for the industrialization of Hypsizigus marmoreus and for the further exploitation of Hypsizigus marmoreus functionality food. The main research output is following:1、The effects of some nutritional factors on the submerged fermentation of Hypsizigus marmoreus were studied in this article. An optimum medium was obtained through single factor tests and optimization tests. And the study on submerged culture of Hypsizigus marmoreus 9603 determined the optimum carbon source and the nitrogen sources. The result showed that potato, glucose, yeast powder were favorable to the mycelium growing of Hypsizigus marmoreus. An optimum medium was obtained by the further orthogonal test. The optimum medium composition of fermentation was as follows: potato 20%, glucose 3%, yeast powder 0.2%,KH₂PO₄ 0.1%, MgS0₄·7H₂O0.1%.2、The submerged culture conditions for Hypsizigus marmoreus 9603 were studied with the method of shake flask. The result showed that the optimum submerged culture conditions for Hypsizigus marmoreus 9603 were listed as follows: the stirring speed at 120 r/min, temperature at 20℃, initial pH at 6.0-7.5（not necessary to regulate）, inoculation volume at about 5ml（250mL erlenmeyer flask, aeration ratio at 100ml）, inoculation quantity 5%.The aeration ratio at 100mL（250mL erlenmeyer flask）. 3、During the culture process of Hypsizigus marmoreus 9603 liquid spawns, the variation of mycelial pellet numbers, pH value, the contents of reducing sugar and amino nitrogen and the enzyme activity were studied. The results showed that the numbers and activity of mycelial pellets were related to those indexes, and that the quality of the liquid spawn was the best when the contents of reducing sugar and amino nitrogen were 0.98g/100mL and 7.52mg/100mL resp, and the activities of cellulase and Proteinase were 7.85U and 12.6U resp. The number（sixty per milliliter） and activity of pellets reached largest when the pH value reached 5.20, and the fermentative period on the 7th day.4、The experiments to compare liquid and solid spawn of Hypsizigus marmoreus and the benefit analysis were carried out. The results showed that contrast to solid spawn culture, the liquid spawn had a lot of advantages such as short cycle, orderly growth and high efficiency. The promising prospect of liquid spawn was proved adequately. The inoculum of 15 mL per hole is the best. During the range of 0-25℃, the viability of liquid spawn of Hypsizigus marmoreus 9603 was minus relative to the temperature and time of storage. The optimal temperature is 0-10℃, The optimal time is 3-9 days. The mycelia of liquid spawn by centesis grew 2.21 mm per day. The mycelia of liquid spawn by surface grew 1.71 mm per day , and the mycelia of solid spawn grew 1.21 mm per day. The biology efficiency of Hypsizigus marmoreus 9603 which was cultivated on liquid spawn by centesis was accounted 64.80% for the dry weight of medium, the corresponding yield of liquid spawn by surface and solid spawn were 63.40% and 51.00%. Contrast to solid spawn culture mode, the fugus growed more orderly by the liquid spawn mode while the other characters were the same.5、The chemical components of the Hypsizigus marmoreus fermentation solution were analyzed. Systematic pre-experiment showed that the fermentation solution of Hypsizigus marmoreus 9603 contained amino acid, protein, polypeptide, organic acid, saponin, inner ester, coumarin, naphtha, hydroxybenzene, sterol and triterpene. Systematic pre-experiment also showed that the fermentation solution of Hypsizigus marmoreus 9603 didn’t contain alkaloid, tan and cyanogen. By method of GC/MS, the chemical components of the volatile gas in fermental solution of Hypsizigus marmoreus 9603 contained alkane, mellow, ketone, aldehyde and other kinds of substance. And mellow （7.46%） and ketone （7.13%） is in the majority. The chemical components in the chloroform extraction of the Hypsizigus marmoreus 9603 fermental solution were determined, too. The result showed that it contained alkane, hydroxybenzene, ester, mellow, ketone and other kinds of substance. And alkane （12.76%） and hydroxybenzene （10.63%） is in the majority. Among these substances, plenty of them have been proved to possess good health care function such as anti-oxidation or anti-caducity function. This research afforded a certain theory foundation and application value for the further exploitation of Hypsizigus marmoreus functionality food.更多还原