真姬菇液体发酵工艺优化及发酵产物分析
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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%、KH_2PO_40.1%、MgSO_4·7H_2O0.1%。
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。
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%,KH_2PO_4 0.1%, MgS0_4·7H_2O0.1%.
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.
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%、KH_2PO_40.1%、MgSO_4·7H_2O0.1%。
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。
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%,KH_2PO_4 0.1%, MgS0_4·7H_2O0.1%.
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.
引文
[1]上宫舟建.真姬菇生物学特性及栽培技术研究[J].中国食用菌,2004,(1):16-17.
[2]郑峻,林友照,胡开辉.真姬菇工厂化栽培关键技术[J].福建农业科技,2006,(8):18-19.
[3]暴增海,马桂珍,肖巧琳.真姬菇的生物学特征及栽培[J].安徽农业科学,2007,35(18):5423,5431.
[4]黄志龙,肖淑霞.真姬菇栽培特性研究[J].福建农业科技,2002,(3):11-12.
[5]程继红,冯志勇.米糠添加量对真姬菇产量和品质的影响[J].食用菌学报,2004,11(1):42-45.
[6]姜华,蔡德华.真姬菇子实体蛋白质营养价值评价[J].食用菌,2005(4):61-62.
[7]刘振伟.真姬菇的特性及其栽培技术[J].中国果菜,2001,(4):15-16.
[8]郑宇,林兴生,陈福如.真姬菇生物学特性研究初报[J].食用菌,2001,(3):12-13.
[9]王新丽.真姬菇Hm-4生物特性及栽培初探[J].食用菌,2002,(1):14-15.
[10]胡开辉,出小平-外界因子对蟹味菇菌丝特性的影响[J].中国食用菌,2008,27(2):16-18.
[11]陆中华,陈俏彪.食用菌贮藏与加工技术[M].北京:中国农业出版社,2004:56-57.
[12]黄清荣,杨立红,刘书涛,等.黄伞液体培养碳氮源的优选[J].吉林农业大学学报,2003,25(6):11-14.
[13]谢小保,谭宏,黄小茉,等.黑曲霉GD-6纤维素酶液体发酵条件的研究[J].微生物学杂志,2004,24(2):21-23.
[14]王耀松,刑增涛,冯志勇,等.真姬菇营养成分的测定与分析[J].菌物研究,2006,4(4):33-37.
[15]乔长晟,汤凤霞.金针菇液体发酵工艺条件的研究[J].宁夏农学院学报,2001,22(2):42-43.
[16]温鲁.食用菌生产技术进展[J].北方园艺,2002,8(1):56-57.
[17]胡开辉,陈体强,黄志龙.食用菌细菌性病害[J].福建农业科技,2000,(3):10-12.
[18]刘传富,皇传华.鸡腿蘑液体菌种培养及应用研究[J].食用菌学报,2004,11(2):20-25.
[19]刘祖同.食用蕈菌生物技术及其应用[M].北京:清华大学出版社,2002:207-229.
[20]辛晓林.食用菌深层发酵及其多糖活性研究进展[J].安徽农业科学,2007,35(15):4622-4625.
[21]杜巍,李元瑞,袁静,等.灰树花液体深层培养条件的研究[J].西北农林科技大学学报(自然科学版),2002,30(1):110-112.
[22]汤亚杰,钟建订.真菌深层发酵生产有用生物活性物质[J].华东理工大学学报,2001,27(6):704-711.
[23]上官舟建,张运茂,林汝楷,等.真姬菇栽培的相关技术研究[J].食用菌,2003,25(6):25-26.
[24]吴金男,吴良友.金针菇液体发酵的营养需求[J].食用菌学报,2003,14(7):41-42.
[25]黄清荣,杨立红,钟旭生,等.真姬菇深层培养碳氮源及无机盐的优选[J].湖北农业科学,2005(6):79-81.
[26]Yuuichi Ukawa,Hitoshi Ito,Makoto Hisamatsu,et al.Antitumor Efects of(1-3)-β-D-Glucan and(1-6)-13-DGlucan Purified from Newly Cultivated Mushroom Hatakeshimeji(Lyophyllu m decmtes Sing).Journal of Bioscience and Bioengineering,2000,90(1):98-104.
[27]吴锦忠,张志强,张丽明,等.虎奶菇化学成分分析[J].福建中医学院学报,2004,1(14):8-10.
[28]李惠珍,黄德鑫,许旭萍,等.正红菇的化学成分的研究[J].菌物系统.1998,17(1):68-74.
[29]张卉.姬松茸发酵液多糖的分离纯化及理化性质分析[J].沈阳化工学院学报,2005,4(19):245-248.
[30]黄清荣,杨立红,刘书涛,等.黄伞液体培养碳氮源的优选[J].吉林农业大学学报,2003,25(6):11-14.
[31]S K Lam,T B Ng.Hypsin a Novel Thcnnostablc Ribosome-Inactivating Protein with ntifLmgai and Antiprolifcrative Activities from Fruiting Bodies of the Edible Mushroom Hypsizigus marmoreus[J].BiochemicaL and Biophysical Research Communications,2001,28(5):1071-1075
[32]Kazntaka Tsnchida,Yutaka Aoyagi,Shoji Odani,et al.Isolation of a Novel Collagen-binding Protein from the Mushroom,Hypsizigus marmoreus,Which Inhibits the Lewis Lung Carcinoma Cell Adhesion to Type Ⅳ Collagcn[J].J Biochem,1995,270(4):1481-1484.
[33]Liotta,L A Rao.Biochemical interactions of tumor cells with the basement membrane[J].Annu Rev Biochem,1986,55:1037-1057.
[34]Yutaka Shoh,Mainorn Iscmn ra,Hamhiko Muto,et al.Isolation of a 41 kDa Protein with Cell Adhesion Activity for Animal Cells from the Mushroom Hypsizigus marmoreus by Affinity Chromatography with Type Ⅳ Collamobilized[J].Biotechnol Biochem,2000,64(4):775-780.
[35]Tetsuro Ikekawa.Beneficial Effects of Edible and Medicinal Mushrooms on Health Care[J].International journal of Medicinal Mushrooms,2001,(3):291-298.
[36]Ikekawa T,Saitoh H,Feng W,et al.Antitumor activity of Hypsizigus marmoreus Ⅰ:Antitumor activity of extracts and polysaccharides[J].Chemical & Pharmaceutical Bulletin,1992,40(7):1954-1957.
[37]Snsnmn Marn yaina.HIV-1 Protease-Inhibiting Substances[J].AIST Today,2002,2(10):11.
[38]Pinto DM,Ning YB,Figeys D.An enhanced microfluidic chip coupled to an electrospray QSTAR mass spectrometer for protein identification[J].Electro phoresis,2000,21(1):181.
[39]Ikemizu Shoichi,Kishimoto Makio,Konishi Haruo.Angiotcnsin-converting enzyme inhibitors of bunashimeji and enokitake[J].Nucleic Acids Res,2000,4(2):451-453.
[40]张建辉,赵经周.食药用菌的液体培养[J].林业科技,2002,27(4):61-63.
[41]黄清荣,辛晓林.真姬菇深层培养条件的研究[J].安徽农业科学,2006,34(3):461-463.
[42]BellarTA,Lichtenberg J J.J Am Water Works Assoc,1974,6(6):739.
[43]黄年来.栽培真姬菇的日本技术[J].中国食用菌,2000,19(6):27.
[44]黄清荣,卜庆梅,刘书涛,等.黄伞液体培养基的正交试验研究[J].江苏农业科学,2004,(2):77-79.
[45]刘冬,李世敏,许柏球,等.灵芝菌丝体深层液体培养基研究[J].微生物学杂志,2001,21(2):15-17.
[46]刘士旺.羊肚菌液体培养基研究初报[J].食用菌学报,1998,5(3):31-37.
[47]林维宣,张水华,董文宾,等.食品分析[M].北京:中国轻工业出版社,1994:25-27.
[48]苏拔贤,范培昌,程明哲,等.生物技术导论[M].北京:人民教育出版社,1978:41-42.
[49]Fan L,Dubois M,Gilles K.Plellulose Hydrolysis[M].Spring-verlag,New York.1997:121-147.
[50]诸葛健,王正祥编著.工业微生物实验技术手册[M].北京:中国轻工业出版社,1997.
[51]B.施特尔马赫(钱嘉渊译).酶的测定方法[M].北京:中国轻工业出版社,1992.8.
[52]姜锡瑞,段钢著.新编酶制剂实用技术手册[M].北京:中国轻工出版社,2002.
[53]谭伟.六种碳源和氮源对长根金钱菌菌丝生长的影响[J].西南农业学报,2001,14(3):109-112.
[54]南京农学院主编.田间试验和统计方法[M].北京:农业出版社,1999:95-97.
[55]康健,王海华.不同浓度MgSO_4和KH_2PO_4对香菇液体培养生长量的影响[J].中国食用菌,2001,20(20):35-37.
[56]宋锡全,刘保东,邱奉同.新编食用菌栽培学[M].赤峰:内蒙古科学技术出版社,2001:296-297.
[57]俞俊棠,唐孝宣.生物工艺学(上)[M].上海:华东理工大学出版社,1999:99-104.
[58]贵州农学院.生物统计学附实验设计[M].北京:农业出版社,1980:183-194.
[59]李松岗.实用生物统计学[M].北京:北京大学出版社,2002:396-399.
[60]张勤,张启能.生物统计学[M].北京:中国农业大学出版社,2002:281-285.
[61]李上彬,曾绩.真姬菇高产栽培技术[J].食用菌,2006,(1):46-47.
[62]秦俊哲,吕嘉析.食用菌贮藏保鲜与加工新技术[M].北京:化学工业出版社,2003:36-38.
[63]贺小贤.生物工艺原理[M].北京:化学工业出版社,2003:48-52.
[64]Katan MB,Zock PL &Mensink RP.Trans fatty acids and their effects on iipoproteins in humans[J].Annual Reviews of Nutrition,1995,15(2):473-493.
[65]S.K.Srivostava and Shri Panjan.Long-term Annual Applications Increase Soil Organic Matter and Nitorgen and Decrease Carbon to Nitorgen Ratio[J].Soil Science Society of America Joumal,1988,52(6):1668-1672.
[66]姚汝华.微生物工程工艺原理[M],广州:华南理工大学出版社,2002:52-53.
[67]乐超银,邵伟.猴头菇液体发酵条件的研究[J].中国食用菌,2002,(3):51-52.
[68]刘祖同,罗信昌.食用覃菌生物技术及应用[M].北京:清华大学出版社,2002:62-65.
[69]丹尼尔.发酵与酶工艺学[M].北京:科学技术出版社,2001:41-48.
[70]王耀松,邢增涛,冯志勇.真姬菇营养成分的测定与分析[J].菌物研究,2006,(4):45-46.
[71]郑集.生物化学[M].北京:高等教育出版社,1997:25-27.
[72]宋爱荣,韩立忠,刘作亭,等.七个白色金针菇菌株发酵液中四种胞外酶活性的测定与分析[J].中国食用菌,1998,18(4):31-34.
[73]尉晓宇.食用菌酶学的应用研究[J].中国食用菌,1999,19(5):15-16.
[74]上官舟建,张运茂,林汝楷,等.真姬菇栽培的相关技术试验[J].食用菌,2003,(6):25-26.
[75]黄清荣,钟旭生,李霞,等.黄伞深层培养条件研究[J].江苏农业科学,2005,(1):97-99.
[76]高连超,韩守玉.用液体菌种栽培杏鲍菇[J].北京农业,2002,23(6):18-19.
[77]王谦,王淑艳.榆黄蘑液体菌种的发酵工艺研究及应用[J].河北农业大学学报,2002,25(1):58-60.
[78]Akira,Seiki G S.Effect of strain and cultivation medium on the chemical composition of the taste components in fruit-body of Hypsizygus marmoreus[J].Food Chemistry,2004, 8(4):265-270.
[79]孙培龙,魏红福,杨井,等.真姬菇研究进展[J].食品科技,2005,(9):54-57.
[80]刘冬,李世敏,许柏球,等.灵芝菌丝体深层液体培养基研究[J].微生物学杂志,2001,21(2):15-17.
[81]陈晓青,蒋新宇.中草药成分分离分析技术与方法[M].北京:化学工业出版社,2006:74-78.
[82]宋小妹,唐志书.中药化学成分提取分离与制备[M].北京:人民卫生出版社,2004:14-17.
[83]叶舟.大叶桉叶精油化学成分及其抑菌活性[J].福建林学院学报,2007,27(1):48-51.
[84]何海斌,陈祥旭,林瑞余,等.化感水稻P1312777苗期根系分泌物中化学成分分析[J].应用生态学报,2005,16(12):2383-2388.
[85]JosephA.Maga.Mushroom Flavor[J].Journal of Agricultural and Food Chemistry,1981,29(1):1-4.
[86]Fountoulakis M,Juranville JF,Roder D.Rreference map of the low molecular mass proteins of haemophilus influence[J].Electrophoresis,1998,19(1):18-19.
[87]ShirleyM.Picard& Phillip issenberg.Investigation of some Volatile constiuents of mushrooms:Changeswhich occurduringheating[J].J.Agric.Foodchem.1973,21(2):959-962.
[88]Kalra,C.L.The chemistryand technologyof amla Phyllanthusemblica resume[J].India Food Packer,2001,42(4):67-82.
[89]邢增涛,郭倩,冯志勇,等.姬松茸中挥发性风味物质的GC-MS分析[J].中药材,2003,26(11):789-791.
[90]Yasukawa Ken,Aoki Takashi,Takido Michio.Inhibitory effects of ergosterol isolated from the edible mushroom Hypsizigus marmoreus on TPA-induced inflammatory earedema and tumor promotion inmice[J].Phytotherapy Research,1994,8(1):10-13.
[91]Assaf S,Hadar Yitzhak,DosoretzCG.1-octen-3-ol and 13-hydroperoxylinoleate are products of distinct pathways in the oxidative breakdown oflinoleic acid by Pleurotus pulmonarius[J].EnzymeMicrobTechnol,1997,21:484-490.
[92]朱效刚,陶冠军,陈蕴.高效液相色谱法测定功能性红曲中的麦角甾醇[J].食品与发酵工业,2005,26(2):104-107.
[93]王尊牛,王升厚,彭勤生.冬虫夏草菌丝体固体发酵粉中麦角甾醇的定量分析[J].沈阳师范大学学报(自然科学版),2005,23(3):293-296.
[94]Tan Tianwei,Zhang Mu,Gao Hua.Ergosterol production by fed-batch fermentation of Saccharomyces cerevisiae[J].Enzyme Microb Technol,2003,33(4):366-369.
[95]Takaku T,KinuraY,Okuda H.Colorimetric method for determination of sugar and related substances[J].Journal of Nutrition,2001,13(1):1409-1413.
[96]Gao Hong,Gu Wenying,Ding Xiaolin.Food Research and D evelopment.2006,6(1):52-54.
[97]郑建仙.功能性食品[M].北京:中国轻工业出版社,1999:49-53.
[98]沈秀荣.金针菇FV908菌株液体培养工艺[J].生物技术,2001,11(6):23-24.
[99]罗韦,李东.香菇多糖的研究进展[J].食品与发酵工业,2000,26(4):63-671.
[2]郑峻,林友照,胡开辉.真姬菇工厂化栽培关键技术[J].福建农业科技,2006,(8):18-19.
[3]暴增海,马桂珍,肖巧琳.真姬菇的生物学特征及栽培[J].安徽农业科学,2007,35(18):5423,5431.
[4]黄志龙,肖淑霞.真姬菇栽培特性研究[J].福建农业科技,2002,(3):11-12.
[5]程继红,冯志勇.米糠添加量对真姬菇产量和品质的影响[J].食用菌学报,2004,11(1):42-45.
[6]姜华,蔡德华.真姬菇子实体蛋白质营养价值评价[J].食用菌,2005(4):61-62.
[7]刘振伟.真姬菇的特性及其栽培技术[J].中国果菜,2001,(4):15-16.
[8]郑宇,林兴生,陈福如.真姬菇生物学特性研究初报[J].食用菌,2001,(3):12-13.
[9]王新丽.真姬菇Hm-4生物特性及栽培初探[J].食用菌,2002,(1):14-15.
[10]胡开辉,出小平-外界因子对蟹味菇菌丝特性的影响[J].中国食用菌,2008,27(2):16-18.
[11]陆中华,陈俏彪.食用菌贮藏与加工技术[M].北京:中国农业出版社,2004:56-57.
[12]黄清荣,杨立红,刘书涛,等.黄伞液体培养碳氮源的优选[J].吉林农业大学学报,2003,25(6):11-14.
[13]谢小保,谭宏,黄小茉,等.黑曲霉GD-6纤维素酶液体发酵条件的研究[J].微生物学杂志,2004,24(2):21-23.
[14]王耀松,刑增涛,冯志勇,等.真姬菇营养成分的测定与分析[J].菌物研究,2006,4(4):33-37.
[15]乔长晟,汤凤霞.金针菇液体发酵工艺条件的研究[J].宁夏农学院学报,2001,22(2):42-43.
[16]温鲁.食用菌生产技术进展[J].北方园艺,2002,8(1):56-57.
[17]胡开辉,陈体强,黄志龙.食用菌细菌性病害[J].福建农业科技,2000,(3):10-12.
[18]刘传富,皇传华.鸡腿蘑液体菌种培养及应用研究[J].食用菌学报,2004,11(2):20-25.
[19]刘祖同.食用蕈菌生物技术及其应用[M].北京:清华大学出版社,2002:207-229.
[20]辛晓林.食用菌深层发酵及其多糖活性研究进展[J].安徽农业科学,2007,35(15):4622-4625.
[21]杜巍,李元瑞,袁静,等.灰树花液体深层培养条件的研究[J].西北农林科技大学学报(自然科学版),2002,30(1):110-112.
[22]汤亚杰,钟建订.真菌深层发酵生产有用生物活性物质[J].华东理工大学学报,2001,27(6):704-711.
[23]上官舟建,张运茂,林汝楷,等.真姬菇栽培的相关技术研究[J].食用菌,2003,25(6):25-26.
[24]吴金男,吴良友.金针菇液体发酵的营养需求[J].食用菌学报,2003,14(7):41-42.
[25]黄清荣,杨立红,钟旭生,等.真姬菇深层培养碳氮源及无机盐的优选[J].湖北农业科学,2005(6):79-81.
[26]Yuuichi Ukawa,Hitoshi Ito,Makoto Hisamatsu,et al.Antitumor Efects of(1-3)-β-D-Glucan and(1-6)-13-DGlucan Purified from Newly Cultivated Mushroom Hatakeshimeji(Lyophyllu m decmtes Sing).Journal of Bioscience and Bioengineering,2000,90(1):98-104.
[27]吴锦忠,张志强,张丽明,等.虎奶菇化学成分分析[J].福建中医学院学报,2004,1(14):8-10.
[28]李惠珍,黄德鑫,许旭萍,等.正红菇的化学成分的研究[J].菌物系统.1998,17(1):68-74.
[29]张卉.姬松茸发酵液多糖的分离纯化及理化性质分析[J].沈阳化工学院学报,2005,4(19):245-248.
[30]黄清荣,杨立红,刘书涛,等.黄伞液体培养碳氮源的优选[J].吉林农业大学学报,2003,25(6):11-14.
[31]S K Lam,T B Ng.Hypsin a Novel Thcnnostablc Ribosome-Inactivating Protein with ntifLmgai and Antiprolifcrative Activities from Fruiting Bodies of the Edible Mushroom Hypsizigus marmoreus[J].BiochemicaL and Biophysical Research Communications,2001,28(5):1071-1075
[32]Kazntaka Tsnchida,Yutaka Aoyagi,Shoji Odani,et al.Isolation of a Novel Collagen-binding Protein from the Mushroom,Hypsizigus marmoreus,Which Inhibits the Lewis Lung Carcinoma Cell Adhesion to Type Ⅳ Collagcn[J].J Biochem,1995,270(4):1481-1484.
[33]Liotta,L A Rao.Biochemical interactions of tumor cells with the basement membrane[J].Annu Rev Biochem,1986,55:1037-1057.
[34]Yutaka Shoh,Mainorn Iscmn ra,Hamhiko Muto,et al.Isolation of a 41 kDa Protein with Cell Adhesion Activity for Animal Cells from the Mushroom Hypsizigus marmoreus by Affinity Chromatography with Type Ⅳ Collamobilized[J].Biotechnol Biochem,2000,64(4):775-780.
[35]Tetsuro Ikekawa.Beneficial Effects of Edible and Medicinal Mushrooms on Health Care[J].International journal of Medicinal Mushrooms,2001,(3):291-298.
[36]Ikekawa T,Saitoh H,Feng W,et al.Antitumor activity of Hypsizigus marmoreus Ⅰ:Antitumor activity of extracts and polysaccharides[J].Chemical & Pharmaceutical Bulletin,1992,40(7):1954-1957.
[37]Snsnmn Marn yaina.HIV-1 Protease-Inhibiting Substances[J].AIST Today,2002,2(10):11.
[38]Pinto DM,Ning YB,Figeys D.An enhanced microfluidic chip coupled to an electrospray QSTAR mass spectrometer for protein identification[J].Electro phoresis,2000,21(1):181.
[39]Ikemizu Shoichi,Kishimoto Makio,Konishi Haruo.Angiotcnsin-converting enzyme inhibitors of bunashimeji and enokitake[J].Nucleic Acids Res,2000,4(2):451-453.
[40]张建辉,赵经周.食药用菌的液体培养[J].林业科技,2002,27(4):61-63.
[41]黄清荣,辛晓林.真姬菇深层培养条件的研究[J].安徽农业科学,2006,34(3):461-463.
[42]BellarTA,Lichtenberg J J.J Am Water Works Assoc,1974,6(6):739.
[43]黄年来.栽培真姬菇的日本技术[J].中国食用菌,2000,19(6):27.
[44]黄清荣,卜庆梅,刘书涛,等.黄伞液体培养基的正交试验研究[J].江苏农业科学,2004,(2):77-79.
[45]刘冬,李世敏,许柏球,等.灵芝菌丝体深层液体培养基研究[J].微生物学杂志,2001,21(2):15-17.
[46]刘士旺.羊肚菌液体培养基研究初报[J].食用菌学报,1998,5(3):31-37.
[47]林维宣,张水华,董文宾,等.食品分析[M].北京:中国轻工业出版社,1994:25-27.
[48]苏拔贤,范培昌,程明哲,等.生物技术导论[M].北京:人民教育出版社,1978:41-42.
[49]Fan L,Dubois M,Gilles K.Plellulose Hydrolysis[M].Spring-verlag,New York.1997:121-147.
[50]诸葛健,王正祥编著.工业微生物实验技术手册[M].北京:中国轻工业出版社,1997.
[51]B.施特尔马赫(钱嘉渊译).酶的测定方法[M].北京:中国轻工业出版社,1992.8.
[52]姜锡瑞,段钢著.新编酶制剂实用技术手册[M].北京:中国轻工出版社,2002.
[53]谭伟.六种碳源和氮源对长根金钱菌菌丝生长的影响[J].西南农业学报,2001,14(3):109-112.
[54]南京农学院主编.田间试验和统计方法[M].北京:农业出版社,1999:95-97.
[55]康健,王海华.不同浓度MgSO_4和KH_2PO_4对香菇液体培养生长量的影响[J].中国食用菌,2001,20(20):35-37.
[56]宋锡全,刘保东,邱奉同.新编食用菌栽培学[M].赤峰:内蒙古科学技术出版社,2001:296-297.
[57]俞俊棠,唐孝宣.生物工艺学(上)[M].上海:华东理工大学出版社,1999:99-104.
[58]贵州农学院.生物统计学附实验设计[M].北京:农业出版社,1980:183-194.
[59]李松岗.实用生物统计学[M].北京:北京大学出版社,2002:396-399.
[60]张勤,张启能.生物统计学[M].北京:中国农业大学出版社,2002:281-285.
[61]李上彬,曾绩.真姬菇高产栽培技术[J].食用菌,2006,(1):46-47.
[62]秦俊哲,吕嘉析.食用菌贮藏保鲜与加工新技术[M].北京:化学工业出版社,2003:36-38.
[63]贺小贤.生物工艺原理[M].北京:化学工业出版社,2003:48-52.
[64]Katan MB,Zock PL &Mensink RP.Trans fatty acids and their effects on iipoproteins in humans[J].Annual Reviews of Nutrition,1995,15(2):473-493.
[65]S.K.Srivostava and Shri Panjan.Long-term Annual Applications Increase Soil Organic Matter and Nitorgen and Decrease Carbon to Nitorgen Ratio[J].Soil Science Society of America Joumal,1988,52(6):1668-1672.
[66]姚汝华.微生物工程工艺原理[M],广州:华南理工大学出版社,2002:52-53.
[67]乐超银,邵伟.猴头菇液体发酵条件的研究[J].中国食用菌,2002,(3):51-52.
[68]刘祖同,罗信昌.食用覃菌生物技术及应用[M].北京:清华大学出版社,2002:62-65.
[69]丹尼尔.发酵与酶工艺学[M].北京:科学技术出版社,2001:41-48.
[70]王耀松,邢增涛,冯志勇.真姬菇营养成分的测定与分析[J].菌物研究,2006,(4):45-46.
[71]郑集.生物化学[M].北京:高等教育出版社,1997:25-27.
[72]宋爱荣,韩立忠,刘作亭,等.七个白色金针菇菌株发酵液中四种胞外酶活性的测定与分析[J].中国食用菌,1998,18(4):31-34.
[73]尉晓宇.食用菌酶学的应用研究[J].中国食用菌,1999,19(5):15-16.
[74]上官舟建,张运茂,林汝楷,等.真姬菇栽培的相关技术试验[J].食用菌,2003,(6):25-26.
[75]黄清荣,钟旭生,李霞,等.黄伞深层培养条件研究[J].江苏农业科学,2005,(1):97-99.
[76]高连超,韩守玉.用液体菌种栽培杏鲍菇[J].北京农业,2002,23(6):18-19.
[77]王谦,王淑艳.榆黄蘑液体菌种的发酵工艺研究及应用[J].河北农业大学学报,2002,25(1):58-60.
[78]Akira,Seiki G S.Effect of strain and cultivation medium on the chemical composition of the taste components in fruit-body of Hypsizygus marmoreus[J].Food Chemistry,2004, 8(4):265-270.
[79]孙培龙,魏红福,杨井,等.真姬菇研究进展[J].食品科技,2005,(9):54-57.
[80]刘冬,李世敏,许柏球,等.灵芝菌丝体深层液体培养基研究[J].微生物学杂志,2001,21(2):15-17.
[81]陈晓青,蒋新宇.中草药成分分离分析技术与方法[M].北京:化学工业出版社,2006:74-78.
[82]宋小妹,唐志书.中药化学成分提取分离与制备[M].北京:人民卫生出版社,2004:14-17.
[83]叶舟.大叶桉叶精油化学成分及其抑菌活性[J].福建林学院学报,2007,27(1):48-51.
[84]何海斌,陈祥旭,林瑞余,等.化感水稻P1312777苗期根系分泌物中化学成分分析[J].应用生态学报,2005,16(12):2383-2388.
[85]JosephA.Maga.Mushroom Flavor[J].Journal of Agricultural and Food Chemistry,1981,29(1):1-4.
[86]Fountoulakis M,Juranville JF,Roder D.Rreference map of the low molecular mass proteins of haemophilus influence[J].Electrophoresis,1998,19(1):18-19.
[87]ShirleyM.Picard& Phillip issenberg.Investigation of some Volatile constiuents of mushrooms:Changeswhich occurduringheating[J].J.Agric.Foodchem.1973,21(2):959-962.
[88]Kalra,C.L.The chemistryand technologyof amla Phyllanthusemblica resume[J].India Food Packer,2001,42(4):67-82.
[89]邢增涛,郭倩,冯志勇,等.姬松茸中挥发性风味物质的GC-MS分析[J].中药材,2003,26(11):789-791.
[90]Yasukawa Ken,Aoki Takashi,Takido Michio.Inhibitory effects of ergosterol isolated from the edible mushroom Hypsizigus marmoreus on TPA-induced inflammatory earedema and tumor promotion inmice[J].Phytotherapy Research,1994,8(1):10-13.
[91]Assaf S,Hadar Yitzhak,DosoretzCG.1-octen-3-ol and 13-hydroperoxylinoleate are products of distinct pathways in the oxidative breakdown oflinoleic acid by Pleurotus pulmonarius[J].EnzymeMicrobTechnol,1997,21:484-490.
[92]朱效刚,陶冠军,陈蕴.高效液相色谱法测定功能性红曲中的麦角甾醇[J].食品与发酵工业,2005,26(2):104-107.
[93]王尊牛,王升厚,彭勤生.冬虫夏草菌丝体固体发酵粉中麦角甾醇的定量分析[J].沈阳师范大学学报(自然科学版),2005,23(3):293-296.
[94]Tan Tianwei,Zhang Mu,Gao Hua.Ergosterol production by fed-batch fermentation of Saccharomyces cerevisiae[J].Enzyme Microb Technol,2003,33(4):366-369.
[95]Takaku T,KinuraY,Okuda H.Colorimetric method for determination of sugar and related substances[J].Journal of Nutrition,2001,13(1):1409-1413.
[96]Gao Hong,Gu Wenying,Ding Xiaolin.Food Research and D evelopment.2006,6(1):52-54.
[97]郑建仙.功能性食品[M].北京:中国轻工业出版社,1999:49-53.
[98]沈秀荣.金针菇FV908菌株液体培养工艺[J].生物技术,2001,11(6):23-24.
[99]罗韦,李东.香菇多糖的研究进展[J].食品与发酵工业,2000,26(4):63-671.