银耳菌液体发酵工艺优化及卵孢菌素鉴定
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摘要
银耳多糖是银耳(Tremella fuciformis)的主要生物活性成分,具有增强免疫,抗肿瘤,抗炎症,降血糖、血脂,抗氧化等功能。通江银耳是四川道地药食两用真菌,是银耳中的上品。但是多年来,通江银耳产量一直未能提高,分析原因在于,自然生长的银耳出耳前菌丝会代谢一种红色或者黄色物质,未代谢红色或黄色物质的菌丝体则不出耳；以及通江银耳的伴生菌菌种退化,选育优良伴生菌工作滞后。采用袋料生产难以控制通江银耳子实体的品质,而现代生物发酵技术既能保证活性成分质量,又能进行工业化生产缩短生产周期,节约成本,是解决通江银耳生产面临问题的一种有效方式。但是对通江银耳菌进行液体深层发酵的研究几乎为零,目前,银耳菌其他种的液体发酵所用培养基成分复杂,不利于产物的后续处理,发酵废液对环境危害也较大。发酵过程中随着培养成分的改变,银耳菌不仅代谢产生多糖,同时也会代谢一种红色化合物。文献报道,在银耳出耳前菌丝会代谢一种红色或者黄色物质的现象,未代谢红色物质的菌丝体则不出耳。因此,银耳菌丝产生的红色物质应为银耳生活史所特有的机制。探讨该物质的产生原因及化学结构,对于研究银耳菌的生长机制具有重要意义。但是,该物质的化学结构及其对银耳出耳的作用未见文献报道。
因此,研究通江银耳液体发酵工艺以及发酵获得的银耳多糖的理化性质、生物活性,以及银耳菌代谢产生红色物质的化学结构和功能,对银耳多糖通过液体发酵工业化生产以及银耳出耳的机制研究具有重要意义。本文以发酵目标产物的量为指标,结合目标产物的活性检测,开展全合成培养基液体培养通江银耳菌,并发酵工艺进行优化和对通江银耳菌液体发酵代谢产生红色物质的分离与结构鉴定等两方面的研究。
一、通江银耳全合成培养基液体发酵工艺优化
在发酵参数筛选结果基础上,对通江银耳全合成培养基液体发酵工艺进行优化,实验结果如下：发酵周期为6d时菌丝体和胞外多糖产量达到最大值；最适菌丝体生长及胞外多糖分泌的碳源分别是葡萄糖和乳糖,复合氮源分别是牛肉膏和酸水解酪蛋白。菌丝体和胞外多糖产量最高的初始pH值分别为pH6.0和pH7.0；菌丝体和胞外多糖产量高的发酵温度为25℃；发酵罐扩大培养最佳转速为150r/min；接种量为10%,菌龄2d。适宜通江银耳菌丝体生长的最佳氨基酸是浓度为0.6%的谷氨酸和甘氨酸(1：1)组合,添加浓度为0.4%的甘氨酸和丝氨酸(1：1)组合,适宜胞外多糖的分泌的最佳氨基酸是浓度为0.6%的谷氨酸和甘氨酸(1：1)的组合。对产菌丝体的最佳工艺进行验证,菌丝体产量达12.32±0.15g/L,胞外多糖的产量达5.94±0.27g/L,菌丝体干重和胞外多糖的产量均比未优化前产量高。
二、银耳菌丝体水溶性多糖和胞外多糖的分离
对发酵获得菌丝体和胞外多糖进行提取分离,测定菌丝体多糖(IPS)和胞外多糖(EPS)的单糖组成和相对分子质量大小。实验结果如下：1、通过响应面法建立了菌丝体多糖提取工艺,模型优化结果是提取温度为89.19℃,料液比1：19.07,提取时间5.29h。2、采用脱蛋白、离子交换层析和分子筛凝胶层析方法,从IPS中分离出2个组分命名为IPS1和IPS2,从EPS中分离出2个组份命名为EPSl和EPS2。经HPLC-RID分析EPS2为单一峰,相对分子质量为420602Da,同时采用PMP柱前衍生的方法测定了所获得的各个组份的单糖组成,IPS1由甘露糖,半乳糖,葡萄糖,鼠李糖组成其摩尔比为93.7：47.3：37.4：1,IPS2由甘露糖,鼠李糖,葡萄糖醛酸,葡萄糖,半乳糖组成,其摩尔比为：45.8：1：6.4：18.9：32.2,EPS1由甘露糖,半乳糖,葡萄糖,鼠李糖组成,其摩尔比为：93.7：47.3：37.1：1,EPS2检测出含有甘露糖,半乳糖,葡萄糖,鼠李糖,葡萄糖醛酸,其摩尔比为：2.4：2.7：6.4：1：33.7。
三、银耳菌代谢产生红色物质—卵孢菌素的分离与鉴定
银耳在由菌丝体向子实体转变的过程中会代谢产生红色物质,分离鉴定该化合物的结构,探讨该化合物在银耳产生子实体过程中的作用,为研究银耳的出耳机制奠定化学物质基础,同时对该化合物进行了生物活性研究以便对其开发利用。利用液体发酵技术筛选银耳菌代谢红色物质的培养条件,采用多次柱层析分离方式获得银耳菌代谢产生的红色物质单体,并对该单体进行X-单晶衍射分析,确定其结构为卵孢菌素。根据卵孢菌素的结构特点以及文献报道,通过动物实验测试了其活性。结果如下：1、首次通过对碳氮源的筛选,找到合适的液体深层发酵的方法使银耳菌代谢产生卵孢菌素,浓度为2%的麦芽糖作为碳源,浓度为0.5%豆粕浸出液作为氮源,发酵2-3d,银耳菌开始代谢卵孢菌素。2、采用D101大孔树脂静态吸附卵孢菌素,结果显示卵孢菌素能够被吸附在树脂上,采用70%的乙醇可将卵孢菌素洗脱下来,洗脱液经过浓缩,低温处理可以获得橙红色晶体。通过X衍射的方法鉴定其结构为卵孢菌素。
四、银耳菌丝体水溶性多糖和胞外多糖及卵孢菌素的活性鉴定
1、体外抗氧化的实验结果表明银耳菌丝体水溶性多糖(IPS)活性高于胞外多糖(EPS)活性,IPS2抗氧化活性高于IPS1,EPS2抗氧化活性高于EPS1,体外抗肿瘤活性实验表明,IPS和EPS粗多糖浓度为50mg/mL时在体外具有较强的抑制HeLa细胞的生长的能力。获得性抑郁模型小鼠行为实验中,胞外多糖(EPS)在浓度为100mg/kg的剂量时能显著降低小鼠悬尾测试实验(TST)和小鼠强迫游泳实验(FST)的一动不动时间,在自主活动的实验中没有明显的精神刺激作用,结果显示通江银耳菌发酵获得胞外多糖(EPS)具有抗抑郁样的活性。2、银耳菌产生的卵孢菌素对获得性抑郁模型小鼠行为影响实验结果表明,50mg/kg和25mg/kg两个剂量下都具有降低TST和FST实验小鼠一动不动的时间,25mg/kg剂量组具有降低兴奋度作用,50mg/kg剂量组没有明显升高自主活的次数,结果显示卵孢菌素具有抗抑郁活性。
Polysaccharide is the main active component of the Tremella fuciformis, which has the function of increasing immunity, lowering blood sugar, anti tumor, antioxidant, etc."Tongjiang"Tremella fuciformis is a edible and medical fungus produced in Sichuan,which is one of the best quality of Tremella fuciformis in china. But over the years, Hypoxylon archeri, a companion fungus of "Tongjiang"7rewella fuciformis is degradation, it is difficult to breed excellent companion fungus. For these reasons the yield of "Tongjiang" Tremella fuciformis can not be increased, it difficult to control the quality of products through the field-cultivation of the fruiting body. Modern bio-fermentation technology provides an efficient way to solve this problem. It can not only keep the quality of active components, but also can shorten the production cycle to save the costs. Currently, the components of liquid medium of Tremella fuciformis is complicated and caused troubles for subsequent proceedings. The fermentation waste water is also harmful to environments. During the concentration of substrate in the fermentation broth changes, a red compound is produced as well as polysaccharide. It is reported that the red or yellow compound is accompanied with the fruiting body of Tremella fuciformis without this compound, there is no fruiting body at all. Unfortunately, as far as we know, the structure and its effects to the fruiting body of Tremella fuciformis are not reported yet. To well understand the fruiting body of Tremella fuciformis so as to improve the industrial application of liquid fermentation, it is important to study the physical-chemical property and bio-activity of the Tremella fuciformis polysaccharides and the structure-property relationship of the red compound produced during the fermentation.
Yield of target product in fermentation broth is main purpose, combined with the activity test of the target product, Optimizate the liquid fermentation process of Tremella fuciformis with defined medium and Tremella fuciformis liquid fermentation metabolism to produce red substances, Isolation and structure identification research.
1. Optimization a chemical defind medium to improve the yield of mycelium and exopolysaccharides of Tremella fuciformis. Select suitable fermentation cycle for Tremella fuciformis mycelium submerged fermentation, the carbon and nitrogen sources, initial pH, fermentation temperature, inoculum volume, cell age and rotate speed in the5L fermenter to optimize expand the cultured.Optimum fermentation period is6days, the best carbon source is glucose, the best nitrogen source for the beef extract production medium conditions optimized for the maximum amount of mycelium12.32±0.15g/L; exopolysaccharides get the maximum yield of5.94±0.27g/L.Optimal submerged culture of the Tremella fuciformis mycelium synthetic medium carbon source is0.2%glucose, nitrogen,0.6%glutamic acid and glycine1:1combination of amino acids, the addition of0.4%glycine and serine1:1combination of amino acids.
2. Separation and Purification water-soluble polysaccharide from the mycelium and fermentation broth of Tremella fuciformis.
Optimization of extraction process of polysaccharide from mycelium was investigated by RSM.The optimum extraction conditions were found to be extraction temperature89.19℃, the liquid-solid ratio19.07, the extraction time of5.29h. After removal of protein, IPS was isolated two components named IPS1and IPS2, EPS was also isolated two component named EPS1and EPS2by ion exchange chromatography and molecular size gel filtration chromatography. EPS2is a single peak by HPLC-RID, the relative molecular mass of420602Da, PMP derivatization and HPLC determination the monosaccharide composition, IPS1was composed by mannose, galactose, glucose, rhamnose, the molar ratio is93.7:47.3:37.4:1IPS2was composed by mannose, rhamnose, glucuronic acid, glucose, galactose in the molar ratio:45.8:1:6.4:18.9:32.2; EPS1was composed by mannose, galactose, glucose, rhamnose, the molar ratio of:93.7:47.3:37.1:1, EPS2was composed by mannose, galactose, glucose, rhamnose, glucuronic acid, the molar ratio is2.4:2.7:6.4:1:33.7.
3. Produced red compound----oosporein was islated and identified from Tremella fuciformis
During mycelium of Tremella fuciformis turn to fruiting body, it produced red compound which was isolated and identified its structure, it's important to reveal the mechanism that mycelium turn to fruiting bodies of Tremella fuciformis, while test the compounds biological activity was useful to develop and utilize. Liquid fermentation technology to select culture conditions to pruduced red compound of Tremella fuciformis, multiple column chromatographic separation mode Tremella fuciformis metabolism of red compound monomer and the monomer was analysis byX-crystal diffraction, to identify its structure was oosporein, According to the structural characteristics of the oosporein and reported in the references, test its activity by animal experiments.The results showed2%maltose as a carbon source,0.5%soybean meal leaching solution as nitrogen source, fermentation time2-3d, Tremella fuciformis began to metabolic oosporein.(2) Optimal purification process of oosporein from Tremella fuciformis by D101macroporous resin adsorption and get compound's crystal. Oosporein can completely was adsorbed by D101 macroporous resin in1h and was eluted by70%ethanol, eluent after enrichment, was staticed at4℃for24h, then oosporein was get crystals. Through the X-ray diffraction detect crystals; the compound's structure identification is oosporein.
4. Bio-activity of water-soluble polysaccharide from mycelia、extracellular polysaccharides and oosporein
The results of in vitro antioxidant activity show that water-soluble polysaccharide (IPS) from mycelium was more effective than the extracellular polysaccharide (EPS) on DPPH scavenging activity. IPS2was more effective than IPS1and EPS2was more effective than EPS1, The concentration of IPS and EPS at50mg/mL have strong inhibition ability for HeLa cells in vitro, Effection of EPS on behavior of depression model mice that showed it has antidepressant-like activity.The mice was irrigated stomach with oosporein at50mg/kg and25mg/kg, TST and FST time reduce, The concentration at25mg/kg lowered the numbers of activity,50mg/kg no significant increases the numbers of activity, it showed that oosporein has antidepressant activities.
因此,研究通江银耳液体发酵工艺以及发酵获得的银耳多糖的理化性质、生物活性,以及银耳菌代谢产生红色物质的化学结构和功能,对银耳多糖通过液体发酵工业化生产以及银耳出耳的机制研究具有重要意义。本文以发酵目标产物的量为指标,结合目标产物的活性检测,开展全合成培养基液体培养通江银耳菌,并发酵工艺进行优化和对通江银耳菌液体发酵代谢产生红色物质的分离与结构鉴定等两方面的研究。
一、通江银耳全合成培养基液体发酵工艺优化
在发酵参数筛选结果基础上,对通江银耳全合成培养基液体发酵工艺进行优化,实验结果如下：发酵周期为6d时菌丝体和胞外多糖产量达到最大值；最适菌丝体生长及胞外多糖分泌的碳源分别是葡萄糖和乳糖,复合氮源分别是牛肉膏和酸水解酪蛋白。菌丝体和胞外多糖产量最高的初始pH值分别为pH6.0和pH7.0；菌丝体和胞外多糖产量高的发酵温度为25℃；发酵罐扩大培养最佳转速为150r/min；接种量为10%,菌龄2d。适宜通江银耳菌丝体生长的最佳氨基酸是浓度为0.6%的谷氨酸和甘氨酸(1：1)组合,添加浓度为0.4%的甘氨酸和丝氨酸(1：1)组合,适宜胞外多糖的分泌的最佳氨基酸是浓度为0.6%的谷氨酸和甘氨酸(1：1)的组合。对产菌丝体的最佳工艺进行验证,菌丝体产量达12.32±0.15g/L,胞外多糖的产量达5.94±0.27g/L,菌丝体干重和胞外多糖的产量均比未优化前产量高。
二、银耳菌丝体水溶性多糖和胞外多糖的分离
对发酵获得菌丝体和胞外多糖进行提取分离,测定菌丝体多糖(IPS)和胞外多糖(EPS)的单糖组成和相对分子质量大小。实验结果如下：1、通过响应面法建立了菌丝体多糖提取工艺,模型优化结果是提取温度为89.19℃,料液比1：19.07,提取时间5.29h。2、采用脱蛋白、离子交换层析和分子筛凝胶层析方法,从IPS中分离出2个组分命名为IPS1和IPS2,从EPS中分离出2个组份命名为EPSl和EPS2。经HPLC-RID分析EPS2为单一峰,相对分子质量为420602Da,同时采用PMP柱前衍生的方法测定了所获得的各个组份的单糖组成,IPS1由甘露糖,半乳糖,葡萄糖,鼠李糖组成其摩尔比为93.7：47.3：37.4：1,IPS2由甘露糖,鼠李糖,葡萄糖醛酸,葡萄糖,半乳糖组成,其摩尔比为：45.8：1：6.4：18.9：32.2,EPS1由甘露糖,半乳糖,葡萄糖,鼠李糖组成,其摩尔比为：93.7：47.3：37.1：1,EPS2检测出含有甘露糖,半乳糖,葡萄糖,鼠李糖,葡萄糖醛酸,其摩尔比为：2.4：2.7：6.4：1：33.7。
三、银耳菌代谢产生红色物质—卵孢菌素的分离与鉴定
银耳在由菌丝体向子实体转变的过程中会代谢产生红色物质,分离鉴定该化合物的结构,探讨该化合物在银耳产生子实体过程中的作用,为研究银耳的出耳机制奠定化学物质基础,同时对该化合物进行了生物活性研究以便对其开发利用。利用液体发酵技术筛选银耳菌代谢红色物质的培养条件,采用多次柱层析分离方式获得银耳菌代谢产生的红色物质单体,并对该单体进行X-单晶衍射分析,确定其结构为卵孢菌素。根据卵孢菌素的结构特点以及文献报道,通过动物实验测试了其活性。结果如下：1、首次通过对碳氮源的筛选,找到合适的液体深层发酵的方法使银耳菌代谢产生卵孢菌素,浓度为2%的麦芽糖作为碳源,浓度为0.5%豆粕浸出液作为氮源,发酵2-3d,银耳菌开始代谢卵孢菌素。2、采用D101大孔树脂静态吸附卵孢菌素,结果显示卵孢菌素能够被吸附在树脂上,采用70%的乙醇可将卵孢菌素洗脱下来,洗脱液经过浓缩,低温处理可以获得橙红色晶体。通过X衍射的方法鉴定其结构为卵孢菌素。
四、银耳菌丝体水溶性多糖和胞外多糖及卵孢菌素的活性鉴定
1、体外抗氧化的实验结果表明银耳菌丝体水溶性多糖(IPS)活性高于胞外多糖(EPS)活性,IPS2抗氧化活性高于IPS1,EPS2抗氧化活性高于EPS1,体外抗肿瘤活性实验表明,IPS和EPS粗多糖浓度为50mg/mL时在体外具有较强的抑制HeLa细胞的生长的能力。获得性抑郁模型小鼠行为实验中,胞外多糖(EPS)在浓度为100mg/kg的剂量时能显著降低小鼠悬尾测试实验(TST)和小鼠强迫游泳实验(FST)的一动不动时间,在自主活动的实验中没有明显的精神刺激作用,结果显示通江银耳菌发酵获得胞外多糖(EPS)具有抗抑郁样的活性。2、银耳菌产生的卵孢菌素对获得性抑郁模型小鼠行为影响实验结果表明,50mg/kg和25mg/kg两个剂量下都具有降低TST和FST实验小鼠一动不动的时间,25mg/kg剂量组具有降低兴奋度作用,50mg/kg剂量组没有明显升高自主活的次数,结果显示卵孢菌素具有抗抑郁活性。
Polysaccharide is the main active component of the Tremella fuciformis, which has the function of increasing immunity, lowering blood sugar, anti tumor, antioxidant, etc."Tongjiang"Tremella fuciformis is a edible and medical fungus produced in Sichuan,which is one of the best quality of Tremella fuciformis in china. But over the years, Hypoxylon archeri, a companion fungus of "Tongjiang"7rewella fuciformis is degradation, it is difficult to breed excellent companion fungus. For these reasons the yield of "Tongjiang" Tremella fuciformis can not be increased, it difficult to control the quality of products through the field-cultivation of the fruiting body. Modern bio-fermentation technology provides an efficient way to solve this problem. It can not only keep the quality of active components, but also can shorten the production cycle to save the costs. Currently, the components of liquid medium of Tremella fuciformis is complicated and caused troubles for subsequent proceedings. The fermentation waste water is also harmful to environments. During the concentration of substrate in the fermentation broth changes, a red compound is produced as well as polysaccharide. It is reported that the red or yellow compound is accompanied with the fruiting body of Tremella fuciformis without this compound, there is no fruiting body at all. Unfortunately, as far as we know, the structure and its effects to the fruiting body of Tremella fuciformis are not reported yet. To well understand the fruiting body of Tremella fuciformis so as to improve the industrial application of liquid fermentation, it is important to study the physical-chemical property and bio-activity of the Tremella fuciformis polysaccharides and the structure-property relationship of the red compound produced during the fermentation.
Yield of target product in fermentation broth is main purpose, combined with the activity test of the target product, Optimizate the liquid fermentation process of Tremella fuciformis with defined medium and Tremella fuciformis liquid fermentation metabolism to produce red substances, Isolation and structure identification research.
1. Optimization a chemical defind medium to improve the yield of mycelium and exopolysaccharides of Tremella fuciformis. Select suitable fermentation cycle for Tremella fuciformis mycelium submerged fermentation, the carbon and nitrogen sources, initial pH, fermentation temperature, inoculum volume, cell age and rotate speed in the5L fermenter to optimize expand the cultured.Optimum fermentation period is6days, the best carbon source is glucose, the best nitrogen source for the beef extract production medium conditions optimized for the maximum amount of mycelium12.32±0.15g/L; exopolysaccharides get the maximum yield of5.94±0.27g/L.Optimal submerged culture of the Tremella fuciformis mycelium synthetic medium carbon source is0.2%glucose, nitrogen,0.6%glutamic acid and glycine1:1combination of amino acids, the addition of0.4%glycine and serine1:1combination of amino acids.
2. Separation and Purification water-soluble polysaccharide from the mycelium and fermentation broth of Tremella fuciformis.
Optimization of extraction process of polysaccharide from mycelium was investigated by RSM.The optimum extraction conditions were found to be extraction temperature89.19℃, the liquid-solid ratio19.07, the extraction time of5.29h. After removal of protein, IPS was isolated two components named IPS1and IPS2, EPS was also isolated two component named EPS1and EPS2by ion exchange chromatography and molecular size gel filtration chromatography. EPS2is a single peak by HPLC-RID, the relative molecular mass of420602Da, PMP derivatization and HPLC determination the monosaccharide composition, IPS1was composed by mannose, galactose, glucose, rhamnose, the molar ratio is93.7:47.3:37.4:1IPS2was composed by mannose, rhamnose, glucuronic acid, glucose, galactose in the molar ratio:45.8:1:6.4:18.9:32.2; EPS1was composed by mannose, galactose, glucose, rhamnose, the molar ratio of:93.7:47.3:37.1:1, EPS2was composed by mannose, galactose, glucose, rhamnose, glucuronic acid, the molar ratio is2.4:2.7:6.4:1:33.7.
3. Produced red compound----oosporein was islated and identified from Tremella fuciformis
During mycelium of Tremella fuciformis turn to fruiting body, it produced red compound which was isolated and identified its structure, it's important to reveal the mechanism that mycelium turn to fruiting bodies of Tremella fuciformis, while test the compounds biological activity was useful to develop and utilize. Liquid fermentation technology to select culture conditions to pruduced red compound of Tremella fuciformis, multiple column chromatographic separation mode Tremella fuciformis metabolism of red compound monomer and the monomer was analysis byX-crystal diffraction, to identify its structure was oosporein, According to the structural characteristics of the oosporein and reported in the references, test its activity by animal experiments.The results showed2%maltose as a carbon source,0.5%soybean meal leaching solution as nitrogen source, fermentation time2-3d, Tremella fuciformis began to metabolic oosporein.(2) Optimal purification process of oosporein from Tremella fuciformis by D101macroporous resin adsorption and get compound's crystal. Oosporein can completely was adsorbed by D101 macroporous resin in1h and was eluted by70%ethanol, eluent after enrichment, was staticed at4℃for24h, then oosporein was get crystals. Through the X-ray diffraction detect crystals; the compound's structure identification is oosporein.
4. Bio-activity of water-soluble polysaccharide from mycelia、extracellular polysaccharides and oosporein
The results of in vitro antioxidant activity show that water-soluble polysaccharide (IPS) from mycelium was more effective than the extracellular polysaccharide (EPS) on DPPH scavenging activity. IPS2was more effective than IPS1and EPS2was more effective than EPS1, The concentration of IPS and EPS at50mg/mL have strong inhibition ability for HeLa cells in vitro, Effection of EPS on behavior of depression model mice that showed it has antidepressant-like activity.The mice was irrigated stomach with oosporein at50mg/kg and25mg/kg, TST and FST time reduce, The concentration at25mg/kg lowered the numbers of activity,50mg/kg no significant increases the numbers of activity, it showed that oosporein has antidepressant activities.
引文
[1]De Baets S, Vandamme E J. Extracellular Tremella polysaccharides:structure, properties and applications[J]. Biotechnology letters.2001,23(17):1361-1366.
[2]Ukai S, Hirose K, Kiho T, et al. Antitumor activity on sarcoma 180 of the polysaccharides from Tremella fuciformis Berk.[J]. Chemical & pharmaceutical bulletin.1972,20(10):2293.
[3]Wasser S. Medicinal mushrooms as a source of antitumor and immunomodulating polysaccharides [J]. Applied Microbiology and Biotechnology.2002,60(3):258-274.
[4]徐碧如.银耳生活史的研究[J].微生物学通报.1980,7：241-242.
[5]刘娟,马爱民,盛桂华,等.银耳二型态细胞差异性的初步研究[J].微生物学通报.2007,34(5)：880-884.
[6]黄年来.银耳菌种生产的原理和方法[J].食用菌.2007,29(1)：25-27.
[7]Liu F, Ooi V, Chang S T. Free radical scavenging activities of mushroom polysaccharide extracts[J]. Life Sciences.1997,60(10):763-771.
[8]谢宝贵,卢启泉,饶永斌,等.人胰岛素基因的人工合成及转化银耳的研究[J].食用菌学报.2007,14(002)：1-14.
[9]朱坚,饶永斌,谢宝贵,等.人乳铁蛋白基因转化银耳的研究[J].热带作物学报.2010(007)：1137-1142.
[10]谢宝贵.银耳遗传转化系统的建立及三个品质基因的转化[D].福建农林大学,2004.
[11]吴大康,张久春.银耳深层发酵条件的研究[J].食品科学.2002,23(1)：64-69.
[12]Cho E J, Oh J Y, Chang H Y, et al. Production of exopolysaccharides by submerged mycelial culture of a mushroom Tremella fuciformis[J]. Journal of biotechnology.2006,127(1):129-140.
[13]袁渊,单麟军,吴尧,等.培养条件对银耳菌丝体及胞外多糖产量的影响[J].食品工业科技.2010(2)：242-244.
[14]丁祥.大型食药用真菌松茸多糖的分离纯化研究[J].西华师范大学学报：自然科学版.2012,32(4)：303-308.
[15]肖丽霞,于洪涛,胡晓松,等.猴头菇多糖的纯化工艺[J].食品科学.2011,32(24).
[16]曲瑾郁,任大明.蛹虫草多糖的化学修饰及体外抗氧化能力[J].食品科学.2011,32(15)：58-61.
[17]孙晓春,颜军,何钢,等.川芎多糖的分离纯化及其单糖组成测定[J].四川农业大学学报.2011,29(1)：56-60.
[18]黄妮,熊双丽,卢飞.绵麦冬多糖的分离纯化及自由基清除活性[J].林产化学与工业.2011,31(1)：68-72.
[19]乔彦茹,罗永煌,周帅,等.灰树花多糖GFP75-2-2B的分离及其对刺激巨噬细胞释放NO的影响[J].食用菌学报.2011,17(4)：48-51.
[20]李健军.柑橘果胶多糖可控性降解和寡糖片段的分离分析及其活性研究[D].西北大学,2011.
[21]Gao Q, Seljelid R, Chen H, et al. Characterisation of acidic heteroglycans from Tremella fuciformis Berk with cytokine stimulating activity[J]. Carbohydrate research.1996,288:135-142.
[22]周爱如,吴彦坤,侯元怡.银耳多糖抗肿瘤作用的研究[J].北京大学学报(医学版).1987,3.
[23]Cheng H H, Hou W C, Lu M L. Interactions of lipid metabolism and intestinal physiology with Tremella fuciformis Berk edible mushroom in rats fed a high-cholesterol diet with or without Nebacitin[J]. Journal of agricultural and food chemistry.2002,50(25):7438-7443.
[24]董志恒,魏春利,曲萌,等.IL-2联合银耳多糖激活的同种脾细胞对肝癌实验性治疗的研究[J].中国免疫学杂志.2004,20(005)：356-359.
[25]董志恒,曲萌.IL-2联合银耳多糖激活的脾细胞对体外肿瘤细胞杀伤作用的研究[J].北华大学学报：自然科学版.2004,5(006)：506-508.
[26]陈飞飞,蔡东联.银耳多糖的主要生物学效用研究进展[J].中西医结合学报.2008,6(8)：862-866.
[27]高小荣,刘培勋.多糖构效关系研究进展[J].中草药.2004,35(2)：229-231.
[28]颜军,郭晓强,邬晓勇,等.银耳多糖的提取及其清除自由基作用[J].成都大学学报：自然科学版.2006,25(1)：35-38.
[29]Vining L C, Kelleher W J, Schwarting A E. Oosporein production by a strain of Beauveria bassiana originally identified as Amanita muscaria[J]. Canadian Journal of Microbiology.1962,8(6):931-933.
[30]Takeshita H, Anchel M. Production of Oosporein and its leuco form by Basidiomycete species[J]. Science.1965,147(3654):152.
[31]Brewer D, Jen W C, Jones G A, et al. The antibacterial activity of some naturally occurring 2, 5-dihydroxy-1,4-benzoquinones[J]. Canadian journal of microbiology.1984,30(8):1068-1072.
[32]Terry B J, Liu W C, Cianci C W, et al. Inhibition of herpes simplex virus type 1 DNA polymerase by the natural product oosporein. [J]. Journal of Antibiotics.1992,45(2):286-288.
[33]Eyal J, Mabud M A, Fischbein K L, et al. Assessment ofBeauveria bassiana Nov. EO-1 strain, which produces a red pigment for microbial control[J]. Applied biochemistry and biotechnology.1994, 44(1):65-80.
[34]Nagaokaa T, Nakatab K, Kounoa K, et al. Antifungal activity of oosporein from an antagonistic fungus against Phytophthora infestans[J]. Zeitschrift fur Naturforschung. C. A journal of biosciences. 2004,59(3-4):302-304.
[35]Strasser H, Abendstein D, Stuppner H, et al. Monitoring the distribution of secondary metabolites produced by the entomogenous fungus Beauveria brongniartii with particular reference to oosporein[J]. Mycological Research.2000,104(10):1227-1233.
[36]Seger C, Erlebach D, Stuppner H, et al. Physicochemical properties of oosporein, the major secreted metabolite of the entomopathogenic fungus Beauveria brongniartii [J]. Helvetica chimica acta. 2005,88(4):802-810.
f37]胡丰林,丁晓娟,杨成,等.一种白僵菌中MAO抑制剂的分离纯化和结构鉴定[J].菌物学报.2006,25(2)：273-277.
[38]Dunlop B W, Nemeroff C B. The role of dopamine in the pathophysiology of depression[J]. Archives of General Psychiatry.2007,64(3):327.
[39]彭卫红,王勇,黄忠乾,等.我国银耳研究现状与存在问题[J].食用菌学报.2005,12(001)：51-56.
[40]李璐,吕俊,毕富勇,等.银耳多糖对肝癌细胞株HepG-2增殖的影响[J].皖南医学院学报.2008,27(5)：320-323.
[41]Chen B. Optimization of extraction of Tremella fuciformis polysaccharides and its antioxidant and antitumour activities in vitro [J]. Carbohydrate Polymers.2010,81(2):420-424.
[42]吕俊,李璐,毕富勇.银耳多糖抑制肝癌HepG-2细胞体外增殖机制的实验研究[J].中国肿瘤.2009,18(005)：413-415.
[43]Ukai S, Hirose K, Kiho T, et al. Antitumor activity on sarcoma 180 of the polysaccharides from Tremella fuciformis Berk.[J]. Chemical & pharmaceutical bulletin.1972,20(10):2293.
[44]Wang J, Flaisher-Grinberg S, Li S, et al. Antidepressant-like effects of the active acidic polysaccharide portion of ginseng in mice[J]. Journal of ethnopharmacology.2010,132(1):65-69.
[45]徐光域,颜军,郭晓强,等.硫酸-苯酚定糖法的改进与初步应用[J][J].食品科学.2005,26(8)：342-346.
[46]Zhu H, Cao C, Zhang S, et al. pH-control modes in a 5-L stirred-tank bioreactor for cell biomass and exopolysaccharide production by Tremella fuciformis spore[J]. Bioresource technology.2011.
[47]Lin C L, Wang C C, Chang S C, et al. Antioxidative activity of polysaccharide fractions isolated from Lycium barbarum Linnaeus[J]. International journal of biological macromolecules.2009,45(2): 146-151.
[48]Xu W, Zhang F, Luo Y B, et al. Antioxidant activity of a water-soluble polysaccharide purified from Pteridium aquilinum[J]. Carbohydrate research.2009,344(2):217-222.
[49]Chen N Y, Lai H H, Hsu T H, et al. Induction of apoptosis in human lung carcinoma A549 epithelial cells with an ethanol extract of Tremella mesenterica[J]. Bioscience, biotechnology, and biochemistry.2008(0):803250836.
[50]Steru L, Chermat R, Thierry B, et al. The automated tail suspension test a computerized device which differentiates psychotropic drugs[J]. Progress in Neuro-Psychopharmacology and Biological Psychiatry.1987,11(6):N1-N671.
[51]Steru L, Chermat R, Thierry B, et al. The tail suspension test a new method for screening antidepressants in mice[J]. Psychopharmacology.1985,85(3):367-370.
[52]黄年来,食用菌.中国银耳生产[M].中国农业出版社,2000.
[53]张汉文.银耳菌种生理性变与生态失控所致病害的研究[J].中国食用菌.1999,18(005)：31-32.
[54]Cryan J F, Mombereau C, Vassout A. The tail suspension test as a model for assessing antidepressant activity:review of pharmacological and genetic studies in mice[J]. Neuroscience & Biobehavioral Reviews.2005,29(4-5):571-625.
[55]Porsolt R D, Bertin A, Jalfre M. Behavioral despair in mice a primary screening test for antidepressants.[J]. Archives internationales de pharmacodynamie et de therapie.1977,229(2):327.
[56]Porsolt R D, Bertin A, Jalfre M. Behavioural despair in rats and mice:strain differences and the effects of imipramine[J]. Eur J Pharmacol.1978,51(3):291-294.
[57]Porsolt R D, Anton G, Blavet N, et al. Behavioural despair in rats a new model sensitive to antidepressant treatments [J]. European journal of pharmacology.1978,47(4):379-391.
[58]Porsolt R D, Bertin A, Blavet N, et al. Immobility induced by forced swimming in rats:effects of agents which modify central catecholamine and serotonin activity[J]. European journal of Pharmacology.1979,57(2):201-210.
[59]De Baets S, Vandamme E J. Extracellular Tremella polysaccharides:structure, properties and applications[J]. Biotechnology letters.2001,23(17):1361-1366.
[60]Guo X, Zou X, Sun M. Optimization of extraction process by response surface methodology and preliminary characterization of polysaccharides from Phellinus igniarius[J]. Carbohydrate Polymers. 2010,80(2):344-349.
[61]颜军,侯贤灯,徐开来.柱前衍生HPLC分析银耳多糖的单糖组成[J].中国测试.2011,37(001)：44-46.
[62]Yui T, Ogawa K, Kakuta M, et al. Chain conformation of a glucurono-xylo-mannan isolated from fruit body of Tremella fuciformis Berk[J]. Journal of Carbohydrate Chemistry.1995,14(2):255-263.
[63]Taniguchi M, Kawaguchi T, Tanaka T, et al. Antimicrobial and respiration-inhibitory activities of oosporein.[J]. Agricultural and biological chemistry.1984,48(4):1065-1067.
[64]Gao Q P, Jiang R Z, Chen H Q, et al. Characterization and cytokine stimulating activities of heteroglycans from Tremella fuciformis[J].Planta Medica-Natural Products and Medicinal Plant Research.1996,62(4):297-302.
[65]Gao Q, Killie M K, Chen H, et al. Characterization and cytokine-stimulating activities of acidic heteroglycans from Tremella fuciformis[J]. Planta Medica-Natural Products and Medicinal Plant Research.1997,63(5):457-460.
[66]Manning R O, Wyatt R D. Comparative toxicity of Chaetomium contaminated corn and various chemical forms of oosporein in broiler chicks[J]. Poultry Science.1984,63(2):251-259.
[67]徐文清,沈秀,周则卫,等.银耳多糖对放射和化学损伤小鼠造血功能的保护作用[J].中草药.2006,37(7)：1057-1058.
[68]王瑞雪,颜军,邬晓勇,等.液体深层发酵条件对银耳菌丝体酸性多糖富集的影响[J].食品科学.2009,30(21)175-177.
[2]Ukai S, Hirose K, Kiho T, et al. Antitumor activity on sarcoma 180 of the polysaccharides from Tremella fuciformis Berk.[J]. Chemical & pharmaceutical bulletin.1972,20(10):2293.
[3]Wasser S. Medicinal mushrooms as a source of antitumor and immunomodulating polysaccharides [J]. Applied Microbiology and Biotechnology.2002,60(3):258-274.
[4]徐碧如.银耳生活史的研究[J].微生物学通报.1980,7：241-242.
[5]刘娟,马爱民,盛桂华,等.银耳二型态细胞差异性的初步研究[J].微生物学通报.2007,34(5)：880-884.
[6]黄年来.银耳菌种生产的原理和方法[J].食用菌.2007,29(1)：25-27.
[7]Liu F, Ooi V, Chang S T. Free radical scavenging activities of mushroom polysaccharide extracts[J]. Life Sciences.1997,60(10):763-771.
[8]谢宝贵,卢启泉,饶永斌,等.人胰岛素基因的人工合成及转化银耳的研究[J].食用菌学报.2007,14(002)：1-14.
[9]朱坚,饶永斌,谢宝贵,等.人乳铁蛋白基因转化银耳的研究[J].热带作物学报.2010(007)：1137-1142.
[10]谢宝贵.银耳遗传转化系统的建立及三个品质基因的转化[D].福建农林大学,2004.
[11]吴大康,张久春.银耳深层发酵条件的研究[J].食品科学.2002,23(1)：64-69.
[12]Cho E J, Oh J Y, Chang H Y, et al. Production of exopolysaccharides by submerged mycelial culture of a mushroom Tremella fuciformis[J]. Journal of biotechnology.2006,127(1):129-140.
[13]袁渊,单麟军,吴尧,等.培养条件对银耳菌丝体及胞外多糖产量的影响[J].食品工业科技.2010(2)：242-244.
[14]丁祥.大型食药用真菌松茸多糖的分离纯化研究[J].西华师范大学学报：自然科学版.2012,32(4)：303-308.
[15]肖丽霞,于洪涛,胡晓松,等.猴头菇多糖的纯化工艺[J].食品科学.2011,32(24).
[16]曲瑾郁,任大明.蛹虫草多糖的化学修饰及体外抗氧化能力[J].食品科学.2011,32(15)：58-61.
[17]孙晓春,颜军,何钢,等.川芎多糖的分离纯化及其单糖组成测定[J].四川农业大学学报.2011,29(1)：56-60.
[18]黄妮,熊双丽,卢飞.绵麦冬多糖的分离纯化及自由基清除活性[J].林产化学与工业.2011,31(1)：68-72.
[19]乔彦茹,罗永煌,周帅,等.灰树花多糖GFP75-2-2B的分离及其对刺激巨噬细胞释放NO的影响[J].食用菌学报.2011,17(4)：48-51.
[20]李健军.柑橘果胶多糖可控性降解和寡糖片段的分离分析及其活性研究[D].西北大学,2011.
[21]Gao Q, Seljelid R, Chen H, et al. Characterisation of acidic heteroglycans from Tremella fuciformis Berk with cytokine stimulating activity[J]. Carbohydrate research.1996,288:135-142.
[22]周爱如,吴彦坤,侯元怡.银耳多糖抗肿瘤作用的研究[J].北京大学学报(医学版).1987,3.
[23]Cheng H H, Hou W C, Lu M L. Interactions of lipid metabolism and intestinal physiology with Tremella fuciformis Berk edible mushroom in rats fed a high-cholesterol diet with or without Nebacitin[J]. Journal of agricultural and food chemistry.2002,50(25):7438-7443.
[24]董志恒,魏春利,曲萌,等.IL-2联合银耳多糖激活的同种脾细胞对肝癌实验性治疗的研究[J].中国免疫学杂志.2004,20(005)：356-359.
[25]董志恒,曲萌.IL-2联合银耳多糖激活的脾细胞对体外肿瘤细胞杀伤作用的研究[J].北华大学学报：自然科学版.2004,5(006)：506-508.
[26]陈飞飞,蔡东联.银耳多糖的主要生物学效用研究进展[J].中西医结合学报.2008,6(8)：862-866.
[27]高小荣,刘培勋.多糖构效关系研究进展[J].中草药.2004,35(2)：229-231.
[28]颜军,郭晓强,邬晓勇,等.银耳多糖的提取及其清除自由基作用[J].成都大学学报：自然科学版.2006,25(1)：35-38.
[29]Vining L C, Kelleher W J, Schwarting A E. Oosporein production by a strain of Beauveria bassiana originally identified as Amanita muscaria[J]. Canadian Journal of Microbiology.1962,8(6):931-933.
[30]Takeshita H, Anchel M. Production of Oosporein and its leuco form by Basidiomycete species[J]. Science.1965,147(3654):152.
[31]Brewer D, Jen W C, Jones G A, et al. The antibacterial activity of some naturally occurring 2, 5-dihydroxy-1,4-benzoquinones[J]. Canadian journal of microbiology.1984,30(8):1068-1072.
[32]Terry B J, Liu W C, Cianci C W, et al. Inhibition of herpes simplex virus type 1 DNA polymerase by the natural product oosporein. [J]. Journal of Antibiotics.1992,45(2):286-288.
[33]Eyal J, Mabud M A, Fischbein K L, et al. Assessment ofBeauveria bassiana Nov. EO-1 strain, which produces a red pigment for microbial control[J]. Applied biochemistry and biotechnology.1994, 44(1):65-80.
[34]Nagaokaa T, Nakatab K, Kounoa K, et al. Antifungal activity of oosporein from an antagonistic fungus against Phytophthora infestans[J]. Zeitschrift fur Naturforschung. C. A journal of biosciences. 2004,59(3-4):302-304.
[35]Strasser H, Abendstein D, Stuppner H, et al. Monitoring the distribution of secondary metabolites produced by the entomogenous fungus Beauveria brongniartii with particular reference to oosporein[J]. Mycological Research.2000,104(10):1227-1233.
[36]Seger C, Erlebach D, Stuppner H, et al. Physicochemical properties of oosporein, the major secreted metabolite of the entomopathogenic fungus Beauveria brongniartii [J]. Helvetica chimica acta. 2005,88(4):802-810.
f37]胡丰林,丁晓娟,杨成,等.一种白僵菌中MAO抑制剂的分离纯化和结构鉴定[J].菌物学报.2006,25(2)：273-277.
[38]Dunlop B W, Nemeroff C B. The role of dopamine in the pathophysiology of depression[J]. Archives of General Psychiatry.2007,64(3):327.
[39]彭卫红,王勇,黄忠乾,等.我国银耳研究现状与存在问题[J].食用菌学报.2005,12(001)：51-56.
[40]李璐,吕俊,毕富勇,等.银耳多糖对肝癌细胞株HepG-2增殖的影响[J].皖南医学院学报.2008,27(5)：320-323.
[41]Chen B. Optimization of extraction of Tremella fuciformis polysaccharides and its antioxidant and antitumour activities in vitro [J]. Carbohydrate Polymers.2010,81(2):420-424.
[42]吕俊,李璐,毕富勇.银耳多糖抑制肝癌HepG-2细胞体外增殖机制的实验研究[J].中国肿瘤.2009,18(005)：413-415.
[43]Ukai S, Hirose K, Kiho T, et al. Antitumor activity on sarcoma 180 of the polysaccharides from Tremella fuciformis Berk.[J]. Chemical & pharmaceutical bulletin.1972,20(10):2293.
[44]Wang J, Flaisher-Grinberg S, Li S, et al. Antidepressant-like effects of the active acidic polysaccharide portion of ginseng in mice[J]. Journal of ethnopharmacology.2010,132(1):65-69.
[45]徐光域,颜军,郭晓强,等.硫酸-苯酚定糖法的改进与初步应用[J][J].食品科学.2005,26(8)：342-346.
[46]Zhu H, Cao C, Zhang S, et al. pH-control modes in a 5-L stirred-tank bioreactor for cell biomass and exopolysaccharide production by Tremella fuciformis spore[J]. Bioresource technology.2011.
[47]Lin C L, Wang C C, Chang S C, et al. Antioxidative activity of polysaccharide fractions isolated from Lycium barbarum Linnaeus[J]. International journal of biological macromolecules.2009,45(2): 146-151.
[48]Xu W, Zhang F, Luo Y B, et al. Antioxidant activity of a water-soluble polysaccharide purified from Pteridium aquilinum[J]. Carbohydrate research.2009,344(2):217-222.
[49]Chen N Y, Lai H H, Hsu T H, et al. Induction of apoptosis in human lung carcinoma A549 epithelial cells with an ethanol extract of Tremella mesenterica[J]. Bioscience, biotechnology, and biochemistry.2008(0):803250836.
[50]Steru L, Chermat R, Thierry B, et al. The automated tail suspension test a computerized device which differentiates psychotropic drugs[J]. Progress in Neuro-Psychopharmacology and Biological Psychiatry.1987,11(6):N1-N671.
[51]Steru L, Chermat R, Thierry B, et al. The tail suspension test a new method for screening antidepressants in mice[J]. Psychopharmacology.1985,85(3):367-370.
[52]黄年来,食用菌.中国银耳生产[M].中国农业出版社,2000.
[53]张汉文.银耳菌种生理性变与生态失控所致病害的研究[J].中国食用菌.1999,18(005)：31-32.
[54]Cryan J F, Mombereau C, Vassout A. The tail suspension test as a model for assessing antidepressant activity:review of pharmacological and genetic studies in mice[J]. Neuroscience & Biobehavioral Reviews.2005,29(4-5):571-625.
[55]Porsolt R D, Bertin A, Jalfre M. Behavioral despair in mice a primary screening test for antidepressants.[J]. Archives internationales de pharmacodynamie et de therapie.1977,229(2):327.
[56]Porsolt R D, Bertin A, Jalfre M. Behavioural despair in rats and mice:strain differences and the effects of imipramine[J]. Eur J Pharmacol.1978,51(3):291-294.
[57]Porsolt R D, Anton G, Blavet N, et al. Behavioural despair in rats a new model sensitive to antidepressant treatments [J]. European journal of pharmacology.1978,47(4):379-391.
[58]Porsolt R D, Bertin A, Blavet N, et al. Immobility induced by forced swimming in rats:effects of agents which modify central catecholamine and serotonin activity[J]. European journal of Pharmacology.1979,57(2):201-210.
[59]De Baets S, Vandamme E J. Extracellular Tremella polysaccharides:structure, properties and applications[J]. Biotechnology letters.2001,23(17):1361-1366.
[60]Guo X, Zou X, Sun M. Optimization of extraction process by response surface methodology and preliminary characterization of polysaccharides from Phellinus igniarius[J]. Carbohydrate Polymers. 2010,80(2):344-349.
[61]颜军,侯贤灯,徐开来.柱前衍生HPLC分析银耳多糖的单糖组成[J].中国测试.2011,37(001)：44-46.
[62]Yui T, Ogawa K, Kakuta M, et al. Chain conformation of a glucurono-xylo-mannan isolated from fruit body of Tremella fuciformis Berk[J]. Journal of Carbohydrate Chemistry.1995,14(2):255-263.
[63]Taniguchi M, Kawaguchi T, Tanaka T, et al. Antimicrobial and respiration-inhibitory activities of oosporein.[J]. Agricultural and biological chemistry.1984,48(4):1065-1067.
[64]Gao Q P, Jiang R Z, Chen H Q, et al. Characterization and cytokine stimulating activities of heteroglycans from Tremella fuciformis[J].Planta Medica-Natural Products and Medicinal Plant Research.1996,62(4):297-302.
[65]Gao Q, Killie M K, Chen H, et al. Characterization and cytokine-stimulating activities of acidic heteroglycans from Tremella fuciformis[J]. Planta Medica-Natural Products and Medicinal Plant Research.1997,63(5):457-460.
[66]Manning R O, Wyatt R D. Comparative toxicity of Chaetomium contaminated corn and various chemical forms of oosporein in broiler chicks[J]. Poultry Science.1984,63(2):251-259.
[67]徐文清,沈秀,周则卫,等.银耳多糖对放射和化学损伤小鼠造血功能的保护作用[J].中草药.2006,37(7)：1057-1058.
[68]王瑞雪,颜军,邬晓勇,等.液体深层发酵条件对银耳菌丝体酸性多糖富集的影响[J].食品科学.2009,30(21)175-177.