高产纤维素酶菌株的筛选及其在青贮饲料中的应用研究
摘要
纤维素是地球上最廉价、最丰富的可再生资源,全世界每年的纤维素及半纤维素的生成量为850亿吨,由于纤维素具有水不溶性的高结晶结构,再把它水解成可利用的葡萄糖相当困难。利用微生物产生的纤维素酶来分解和转化纤维素则是其利用的有效途径。目前人们对纤维素酶的研究多集中于霉菌,但霉菌多为好氧性微生物,而青贮饲料发酵是在少氧或缺氧环境中进行的,故筛选产纤维素酶活高的厌氧及兼性厌氧细菌更具有应用上的现实意义。
本研究从自然界野牦牛粪中分离出三株高产纤维素酶细菌菌株YMN8、YMN30、YMN41,经鉴定结果三株菌为芽孢杆菌属细菌,YMN8为短小芽孢杆菌(Bacillus pumilus)、YMN30为多粘芽孢杆菌(Bacillus polymyxa)、YMN41为凝结芽孢杆菌(Bacillus coagulans)。DNS法测纤维素酶活,单一培养后的纤维素酶活分别为3752U/ml、3910U/ml和3975U/ml;经拮抗试验证明这三株细菌菌株间无拮抗作用,组合培养后纤维素酶活有明显提高,最后确定三组合培养其纤维素酶活达到最高为4210U/ml。通过正交试验结果得知,接种量对产酶的影响较大,氮的含量和水的含量影响较小,从而确定了三组合细菌菌剂接种量在0.1%时产纤维素酶活最高。同时,实验确定了将筛选出来的高产纤维素酶细菌与乳酸菌、酵母菌的微生物固体菌剂按1:2:2的混合配比,加入0.5%的尿素,添加到全株玉米青贮原料中,发酵30天后,感官评定为1级优良;与对照组相比,其中干物质(DM)含量提高了11.67%,粗蛋白(CP)含量提高了16.2%,中性洗涤纤维(NDF)和酸性洗涤纤维(ADF)的含量分别降低了16.63%和15.85%,PH为3.54。
通过奶牛喂养试验,对30头中国荷斯坦泌乳牛在相同的饲养条件下进行了30天饲养对比试验。与对照组相比试验组平均日产奶量增加了3.85%。此外,试验组所产牛奶的各种乳指标也均有提高,与对照组相比乳脂含量增加2.51%、乳蛋白含量增加6.40%、乳糖含量增加2.93%,同时提高了试验组奶牛的免疫抗疾病能力。
Cellulose is the cheapest and the most abundant regeneration resource on the earth.the create capacity of the cellulose and hemicellulose is reach to 85 billion ton. Because of the cellulose has the high crystal structural which have water insolubility, it is very difficult to translate for available Glucose. Using the cellulase which produced by the microbiology to decompound and translate the cellulose is an efficacious way. at present people on the research of cellulase are mostly concentrated on the Mildew, but the most of Mildew is the microbiology which need the oxygen and the silage fermentation is going on the condition of little or no oxygen, so screening the high producing cellulase bacteria which the growing course is not need or little need the oxygen have the big practical significance on the application.
The study have screened three bacteria strains YMN8 YMN30 YMN41 from the feces of wild yak on the nature, after the identify the result was that the three stains were belonged to the bacillus bacteria, YMN8 was Bacillus pumilus, YMN30 was Bacillus polymyxa and YMN41 was Bacillus coagulans. Using the way of DNS measure the activity of the cellulase, the single bacteria stain incubate the CMCase activity were reach to 3752U/ml, 3910U/ml and 3975U/ml respectively. The antagonism test proved that between of the bacteria strains hadn't the antagonism action. Two of the strains mixed could improve the cellulase produce, and the three bacteria stains mixed made the highest cellulase activity amounted to 4210U/ml. The orthogonal experiment showed that the inoculation capacity of the mixed strains was the primary element to affect the cellulase activity, the compound of the nirtrogenous and the water was weaker. So the highest cellulase activity produced with the 0.1 percent inoculated. At the same time, the experiment was made the culture proportion of the screened bacteria stains, lactobacillus culture and microzyme culture 1:2:2, the recruitment of urea was 0.5 percent, add to the whole-corn silage with the processing of the micro-fermentation for 30 days. After the fermentation the sensory of the silage was 1 degree best. Compared with the contrast groups, the dry matter (DM) increased by 11.67 percent, the crude protein (CP) increased by 16.2 percent, the neutral detergent fiber (NDF) and the acid detergent fiber (ADF) decreased by 16.63 percent and 15.85 percent, PH was 3.54.
Through the cow feed test with the corn silage, in which the 30 Holstein dairy cattle were conducted with feed comparison trial for 30 days. Compared with the contrast groups, the per day per dairy cow milk production of the test groups increase 3.85 percent, in addition, the every milk containing guideline of test groups were all better than the contrast groups, the milk fat content was increased by 2.51 percent, milk protein was increased by 6.40 percent, lactose was increased by 2.93 percent,at the same time, the test crow's immunity and the ability of resist to illness would be improved.
本研究从自然界野牦牛粪中分离出三株高产纤维素酶细菌菌株YMN8、YMN30、YMN41,经鉴定结果三株菌为芽孢杆菌属细菌,YMN8为短小芽孢杆菌(Bacillus pumilus)、YMN30为多粘芽孢杆菌(Bacillus polymyxa)、YMN41为凝结芽孢杆菌(Bacillus coagulans)。DNS法测纤维素酶活,单一培养后的纤维素酶活分别为3752U/ml、3910U/ml和3975U/ml;经拮抗试验证明这三株细菌菌株间无拮抗作用,组合培养后纤维素酶活有明显提高,最后确定三组合培养其纤维素酶活达到最高为4210U/ml。通过正交试验结果得知,接种量对产酶的影响较大,氮的含量和水的含量影响较小,从而确定了三组合细菌菌剂接种量在0.1%时产纤维素酶活最高。同时,实验确定了将筛选出来的高产纤维素酶细菌与乳酸菌、酵母菌的微生物固体菌剂按1:2:2的混合配比,加入0.5%的尿素,添加到全株玉米青贮原料中,发酵30天后,感官评定为1级优良;与对照组相比,其中干物质(DM)含量提高了11.67%,粗蛋白(CP)含量提高了16.2%,中性洗涤纤维(NDF)和酸性洗涤纤维(ADF)的含量分别降低了16.63%和15.85%,PH为3.54。
通过奶牛喂养试验,对30头中国荷斯坦泌乳牛在相同的饲养条件下进行了30天饲养对比试验。与对照组相比试验组平均日产奶量增加了3.85%。此外,试验组所产牛奶的各种乳指标也均有提高,与对照组相比乳脂含量增加2.51%、乳蛋白含量增加6.40%、乳糖含量增加2.93%,同时提高了试验组奶牛的免疫抗疾病能力。
Cellulose is the cheapest and the most abundant regeneration resource on the earth.the create capacity of the cellulose and hemicellulose is reach to 85 billion ton. Because of the cellulose has the high crystal structural which have water insolubility, it is very difficult to translate for available Glucose. Using the cellulase which produced by the microbiology to decompound and translate the cellulose is an efficacious way. at present people on the research of cellulase are mostly concentrated on the Mildew, but the most of Mildew is the microbiology which need the oxygen and the silage fermentation is going on the condition of little or no oxygen, so screening the high producing cellulase bacteria which the growing course is not need or little need the oxygen have the big practical significance on the application.
The study have screened three bacteria strains YMN8 YMN30 YMN41 from the feces of wild yak on the nature, after the identify the result was that the three stains were belonged to the bacillus bacteria, YMN8 was Bacillus pumilus, YMN30 was Bacillus polymyxa and YMN41 was Bacillus coagulans. Using the way of DNS measure the activity of the cellulase, the single bacteria stain incubate the CMCase activity were reach to 3752U/ml, 3910U/ml and 3975U/ml respectively. The antagonism test proved that between of the bacteria strains hadn't the antagonism action. Two of the strains mixed could improve the cellulase produce, and the three bacteria stains mixed made the highest cellulase activity amounted to 4210U/ml. The orthogonal experiment showed that the inoculation capacity of the mixed strains was the primary element to affect the cellulase activity, the compound of the nirtrogenous and the water was weaker. So the highest cellulase activity produced with the 0.1 percent inoculated. At the same time, the experiment was made the culture proportion of the screened bacteria stains, lactobacillus culture and microzyme culture 1:2:2, the recruitment of urea was 0.5 percent, add to the whole-corn silage with the processing of the micro-fermentation for 30 days. After the fermentation the sensory of the silage was 1 degree best. Compared with the contrast groups, the dry matter (DM) increased by 11.67 percent, the crude protein (CP) increased by 16.2 percent, the neutral detergent fiber (NDF) and the acid detergent fiber (ADF) decreased by 16.63 percent and 15.85 percent, PH was 3.54.
Through the cow feed test with the corn silage, in which the 30 Holstein dairy cattle were conducted with feed comparison trial for 30 days. Compared with the contrast groups, the per day per dairy cow milk production of the test groups increase 3.85 percent, in addition, the every milk containing guideline of test groups were all better than the contrast groups, the milk fat content was increased by 2.51 percent, milk protein was increased by 6.40 percent, lactose was increased by 2.93 percent,at the same time, the test crow's immunity and the ability of resist to illness would be improved.
引文
[1]刘德海,王红云等.纤维素酶高产菌株的选育及其生物性状的研究[J].饲料工业,2005,26(18):24.
[2]王景林等.高活力纤维素酶菌株康氏木霉B-7的选育与产酶条件的研究[J].生物技术,1996,6(6):14-17.
[3]肖春玲.微生物纤维素酶的应用研究[J].微生物学杂志,2002,20(3):10.
[4](日)荒开基夫.纤维素酶研究动向.日本酿造协会杂志,1988;(3)177-182.
[5]Stemberg,D.et al.Appl,Environ.Microbiol.1976,21:648-652.
[6]Stemberg,D,et al.Can.J.Microbil.1977,23:139-147.
[7]Bamburg,J.K.et al.Microbiol Toxins,Academic.Press.1971,17:207-289.
[8]Joffe,A.Z.Microbiol Toxins,Alimentary Toxic Aleukia,in Kadis,S.et al.(Eds.)Academic.Press.1971,7:139-187.
[9]Bisaria.V S and Ghose,T.K.Biodegradation of cellulosic materials:substrates Microorganisms,Microorganisms,enaymes and products.Enzyme Microb.TechnoI,1981,(3):90-104.
[10]艾云灿,赵学慧,余家林.判断纤维素酶系基因组融合重组的统计分析法[J].生物工程学报,1994,10(1):45.
[11]张海,晋神贞.秸秆类农业废弃物的生物利用:Ⅱ秸秆的现代利用方法.农牧产品开发,1995,(6):21-25.
[12]Wood T.M et al.Biochemistry and Genetics of Cellulose Degredation.FEMs symp.London[J].Academic,1988:31.
[13]窦全林,陈刚.纤维素酶的研究进展及应用前景[J].畜牧与饲料科学,2006,5:58-60.
[14]陈阿娜,汤斌.纤维素酶高产菌株选育研究进展[J].安徽农学通报,2006,12(10):64.
[15]赵荣乐.纤维素酶研究进展[J].喀什师范学院学报,2005,26(6):51-54.
[16]王仁振.秸杆类粗纤维饲料生化加工技术的研究[J].饲料工业,1999,20(3):8-12
[17]Wilson,et al.Bioresource Technology,1996,6-7.
[18]Gilkes N,R.,Henrissat B Kilbum D.G.,Miller R.C.Warren R.A.J.Domains in microbial β-1,4-glycanases:sequence conservation,function and enzyme families.[J]microbiol.Rev,1991,55:303-315.
[19]高培基,曲音波等.微生物降解纤维素机制的分子生物学进展.纤维素科学与技术.1995,3(2):1-19.
[20]Gibbs M D,RA Reeves and G K Fairington.Multidomin and multifunctional glycosyl hydrolases from the extrem thermophile Caldicellulosiruptor isolate Tok7B.1[J].curr.Microbiol.2000.40:333-340.
[21]张树政主编.酶制剂工业[M].科学出版社,1984,7.
[22]Ghose TK(1987)Measurement of cellulase activities[J].Pure Appl.Chen.59:257-268.
[23]Katalin Urbanszki,Gyorgy Szakacs,Robert P.Tengerdy.Standardization of the filter paper activity assay for solid substrate fermentation[J].Biotechnolgy Letters,2000,22:65-69.
[24]吴显荣,穆小民.纤维素酶分子生物学研究进展及趋向[J].生物工程进展,1994,14(4):25-27.
[25]宋波,施雷霆.纤维素酶的研究进展[J].上海环境科学,2003,22(7):491-495.
[26]Mandels.Mand E.T.Reese.Induction of cellulase in Trichoderma viride by canbon sources and metals[J].Bacterial.1977:73-269.
[27]王冬,曲音波等.腺昔三磷酸和环腺昔单磷酸对丝状真菌纤维素酶合成的调节[J].微生物学报,1996,36(1):12-18.
[28]汪天虹,王春卉.高培基.纤维素酶纤维素吸附区的结构和功能[J].生物工程进展,2000,20(2):37-40.
[29]Reese E.T.et al.J.Bacterial[M].1950(59):485-497.
[30]Heorissat B.et al.Bio/Tech[M].1985(3):722-726.
[31]Wood T M.et al.Biochemistry and ceneties of centies of celluloseDegredation.FE Ms symp,43.London[J].Academic.1988:21.
[32]Moloney A P.et al.Biochem[J].1985,225(3):365-373.
[33]Reese E T.Biotechnol Bioeng Symposium[J].1981(523):147-161.
[34]Wood T M.The cellulose of fusarium solani purification and specificity of the β-(1,4)-glucosidase components[J].Biochem,1971:121-353.
[35]Faterstam L T et al.FEBS Lett[J].1980(119):97-100.
[36]Kanda T et al.J Biochem[J].1976(79):997-1006.
[37]欧阳平凯.植物纤维素原料的水解及应用[M].第1版.北京:中国科学技术出版社,1995.
[38]Coughlan M.P.细菌和真菌降解纤维素的机理.国外畜牧科技.1991,18(6):2831.
[39]Park.Jin Won.Development of effective modified cellulase for cellulose hydrolysis process.Biotechnoiogy and Bioengineering,1995,45(4):366-373.
[40]Melander.M.Determination of the degree of polymerization of carboxymethyl cellulose by size exclusion chromatography.Carbohydrate Polymers.2001,46(3):227-233.
[41]张元琴,黄勇.以纤维素材料为基质的生物降解材料的研究进展.高分子材料科学与工程,1999,15(5):25-29.
[42]Ramos L.P.Nazhad M.and Saddler J.N.Cffect of enzymatic hydrolysis on the morphology and fine structure of pretreated cellulosic residues.Enzyme Microb.Technol,1993.15:821-831.
[43]刘小杰,何国庆,陈启和.康宁木霉ZJ5纤维素发酵培养基的优化[J].浙江大学学报(工学版),2003,37(5):623-628.
[44]Gao P J,Qu Y B,Zhao X,et al.Screening microbial strain for improving the nutritional value of wheat and corn straws as animal feed.Enzyme Microb Technol,1997,20:581-584.
[45]郑亚平,余晓斌.碳源对绿色木霉ZC产中性纤维素酶的影响[J].无锡轻工大学学报,2003,22(2):30-33.
[46]宋国安.纤维素酶的应用开发前景[J].河南化工,1999,6(5):32.
[47]扬雪霞,陈洪章,李佐虎.添加纤维素酶的青贮研究进展[J].生物技术通报,2001(1):37-38.
[48]王建兵,范石军,刘静波等.细菌接种剂和酶制剂在秸秆类饲料青贮中的应用[J].饲料博览,1999,11(4):17-19.
[49]周德宝.青贮饲料的研究、发展及现状[J].氨基酸和生物资源,2004,(2):32-34.
[50]Wilkinson J M,Stark B.Silage in Westem Europe[C].A Survey of 17 Countries.Marlow,UK:Chalcombe Publications,1987.
[51]李培庆,崔艳红,胡海霞.生物技术在提高秸秆饲料利用率上的研究与应用[J].安徽农业科学,2006,34(10):2074-2076.
[52]王富生,马俊孝,任红卫.微生物青贮剂在玉米秸秆青贮中的作用[J].山东大学学报,2004,39(2):112-115.
[53]杨志刚,沈益新,陈阿琴.纤维素酶在青贮饲料的应用[J].饲料博览2002(1):39-41.
[54]丁健,贾亚红,陈小莲等.纤维素酶对玉米青贮饲料糖和有机酸生成量的影响[J].饲料工业,2002,23(9):10-11.
[55]王安,张淑芳,钟一民.纤维素复合酶作为青贮饲料添加剂的研究[J].东北农业大学学报.1997,28(4):358-365.
[56]史占全,刘建新,张箐桦.添加酶制剂对青贮玉米秸发酵品质和化学成分的影响[J].浙江农业大学学报,1998,24(6):653-657.
[57]冯怀蓉,龙涛,崔卫东等.利用青贮复合菌剂制作青贮饲料的效果测定[J].新疆农业科学,2006,43(1):75-77.
[58]李新胜,张春喜,孙哲等.加酶高粱青贮对奶牛产奶量的影响[J].中国奶牛,2001(1):26-27.
[59]顾锡慧,杨翔华,马学良.青贮添加剂研究进展[J].辽宁石油化工大学学报,2004,24(3):29-30.
[60]杨新刚,沈益新.纤维素酶制剂在青贮饲料中的应用[J].畜牧与兽医,2002(9):37-39.
[61]杨连玉,众岛芳也.化学和生物处理对玉米秸秆营养价值的影响[J].吉林农业大学学报,2001(1):83-87.
[62]常巧玲,孙建议等.纤维素酶对奶牛营养的研究进展[J].中国饲料,2005(1):18-20.
[63]McAllan A B,Jacobs J L,Merry R J.Factors influencing the amount and pattemof silage effluent production[C].Forage Conservation towards 2000 Braunschweig,1991,368-370.
[64]WANG Z Y,ZHANG J D,Li Y.Liquid medium on cordyceps militaris[J].Joumal of Liaoning of petroleum & chemical technology,2004,24(1):133-138.
[65]张海,左玉萍,利用酶制剂作为青贮饲料添加剂[J].草与畜,1996(1):30-31.
[66]郭金双,赵光永,冯仰廉等.纤维素酶对大麦青贮品质及中型洗涤纤维瘤胃降解率的影响[J].饲料工业,2005,26(3):13-14.
[67]王国仓,李增辉,范秀兰.微生物在青贮饲料中的作用[J].内蒙古畜牧科学,2003(3):55-56.
[68]蔡义民,熊井清雄,廖芷.乳酸菌制剂对青贮饲料发酵品质的改善效果[J].中国农业科学,1995,28,(2):73-82.
[69]国春艳,杜红芳,刁其玉等.乳酸菌的生理功能及在畜牧业中的应用[J].饲料工业,2006,27(2):55-57.
[70]周淑芹,孙文志.酵母培养物[J].中国饲料,2003(5):31-32.
[71]杨富裕,张蕴薇.尿素在青贮料中的应用[J].中国奶牛,2005(5):23-24.
[72]郭松林.纤维素酶及其在畜禽生产中的应用[J].中国饲料,2001,9:23-24.
[73]陈侠甫.应用木10纤维素酶治疗马牛胃肠病[J].中国兽医杂志,1988,(2):32-33.
[74]王贵权等.纤维素酶治疗牛消化道疾病150例.黑龙江畜牧兽医.1991,7:31.
[75]只兴有.纤维素酶治疗奶牛低酸度酒精阳性乳[J].黑龙江畜牧兽医,1991,6:33.
[76]周正红,高孔荣等.纤维素酶在食品发酵工业中的应用及前景.暨南大学学报(自科与医学版).1998,19(5):125-130.
[77]邱立友,朱德育.黑曲霉酶学特性及其在洒精生产中的应用.河南农业大学学报,1993,27(3):91-295.
[78]汪多仁.纤维素酶在纺织工业中的应用[J].四川化工与腐蚀控制,2000,5(3):52-53.
[79]洪洞,黄秀梨.纤维素酶的应用[J].生物学通报,1997,32(12):19.
[80]叶姜瑜.一种纤维素分解鉴别培养基[J].微生物学通报,1997,24(4):251-252.
[81]沈雪亮,夏黎明.产纤维素酶细菌的筛选及酶学特性研究[J].林产化学与工业,2002,22(1);47-48.
[82]张虹等.纤维素酶产生菌的分离和鉴定[J].齐齐哈尔师范学院学报(自然科学 版),1997,17(4):62.
[83]中国科学院微生物研究所细菌分类组.一般细菌常用鉴定方法[M].北京:科学出版社,1978.
[84]东方珠,蔡妙英等编著.常见细菌系统鉴定手册[M].北京:科学出版社,2001.
[85]钱嘉渊译.酶的测定方法.北京:中国轻工业出版社,1992.
[86]中国科学院上海植物生理研究所.二株高活力纤素分解菌N2-78和EA3-867的获得及其特性的比较[J].微生物学报,1978,18(1):27-38.
[87]王淑军等.纤维素酶酶解稻壳条件试验[J].微生物学报,1995,6:354-357.
[88]WANG ZY,ZHANG J D,Li Y.Liquid medium on cordyceps militaris[J].Journal of Liaoning o f petroleum & chemical technology,2004,24(1):133-138.
[89]R.E.布坎南,N.E.吉本斯等编著.伯杰细菌鉴定手册(第八版)[M].北京:科学出版社,1984.
[2]王景林等.高活力纤维素酶菌株康氏木霉B-7的选育与产酶条件的研究[J].生物技术,1996,6(6):14-17.
[3]肖春玲.微生物纤维素酶的应用研究[J].微生物学杂志,2002,20(3):10.
[4](日)荒开基夫.纤维素酶研究动向.日本酿造协会杂志,1988;(3)177-182.
[5]Stemberg,D.et al.Appl,Environ.Microbiol.1976,21:648-652.
[6]Stemberg,D,et al.Can.J.Microbil.1977,23:139-147.
[7]Bamburg,J.K.et al.Microbiol Toxins,Academic.Press.1971,17:207-289.
[8]Joffe,A.Z.Microbiol Toxins,Alimentary Toxic Aleukia,in Kadis,S.et al.(Eds.)Academic.Press.1971,7:139-187.
[9]Bisaria.V S and Ghose,T.K.Biodegradation of cellulosic materials:substrates Microorganisms,Microorganisms,enaymes and products.Enzyme Microb.TechnoI,1981,(3):90-104.
[10]艾云灿,赵学慧,余家林.判断纤维素酶系基因组融合重组的统计分析法[J].生物工程学报,1994,10(1):45.
[11]张海,晋神贞.秸秆类农业废弃物的生物利用:Ⅱ秸秆的现代利用方法.农牧产品开发,1995,(6):21-25.
[12]Wood T.M et al.Biochemistry and Genetics of Cellulose Degredation.FEMs symp.London[J].Academic,1988:31.
[13]窦全林,陈刚.纤维素酶的研究进展及应用前景[J].畜牧与饲料科学,2006,5:58-60.
[14]陈阿娜,汤斌.纤维素酶高产菌株选育研究进展[J].安徽农学通报,2006,12(10):64.
[15]赵荣乐.纤维素酶研究进展[J].喀什师范学院学报,2005,26(6):51-54.
[16]王仁振.秸杆类粗纤维饲料生化加工技术的研究[J].饲料工业,1999,20(3):8-12
[17]Wilson,et al.Bioresource Technology,1996,6-7.
[18]Gilkes N,R.,Henrissat B Kilbum D.G.,Miller R.C.Warren R.A.J.Domains in microbial β-1,4-glycanases:sequence conservation,function and enzyme families.[J]microbiol.Rev,1991,55:303-315.
[19]高培基,曲音波等.微生物降解纤维素机制的分子生物学进展.纤维素科学与技术.1995,3(2):1-19.
[20]Gibbs M D,RA Reeves and G K Fairington.Multidomin and multifunctional glycosyl hydrolases from the extrem thermophile Caldicellulosiruptor isolate Tok7B.1[J].curr.Microbiol.2000.40:333-340.
[21]张树政主编.酶制剂工业[M].科学出版社,1984,7.
[22]Ghose TK(1987)Measurement of cellulase activities[J].Pure Appl.Chen.59:257-268.
[23]Katalin Urbanszki,Gyorgy Szakacs,Robert P.Tengerdy.Standardization of the filter paper activity assay for solid substrate fermentation[J].Biotechnolgy Letters,2000,22:65-69.
[24]吴显荣,穆小民.纤维素酶分子生物学研究进展及趋向[J].生物工程进展,1994,14(4):25-27.
[25]宋波,施雷霆.纤维素酶的研究进展[J].上海环境科学,2003,22(7):491-495.
[26]Mandels.Mand E.T.Reese.Induction of cellulase in Trichoderma viride by canbon sources and metals[J].Bacterial.1977:73-269.
[27]王冬,曲音波等.腺昔三磷酸和环腺昔单磷酸对丝状真菌纤维素酶合成的调节[J].微生物学报,1996,36(1):12-18.
[28]汪天虹,王春卉.高培基.纤维素酶纤维素吸附区的结构和功能[J].生物工程进展,2000,20(2):37-40.
[29]Reese E.T.et al.J.Bacterial[M].1950(59):485-497.
[30]Heorissat B.et al.Bio/Tech[M].1985(3):722-726.
[31]Wood T M.et al.Biochemistry and ceneties of centies of celluloseDegredation.FE Ms symp,43.London[J].Academic.1988:21.
[32]Moloney A P.et al.Biochem[J].1985,225(3):365-373.
[33]Reese E T.Biotechnol Bioeng Symposium[J].1981(523):147-161.
[34]Wood T M.The cellulose of fusarium solani purification and specificity of the β-(1,4)-glucosidase components[J].Biochem,1971:121-353.
[35]Faterstam L T et al.FEBS Lett[J].1980(119):97-100.
[36]Kanda T et al.J Biochem[J].1976(79):997-1006.
[37]欧阳平凯.植物纤维素原料的水解及应用[M].第1版.北京:中国科学技术出版社,1995.
[38]Coughlan M.P.细菌和真菌降解纤维素的机理.国外畜牧科技.1991,18(6):2831.
[39]Park.Jin Won.Development of effective modified cellulase for cellulose hydrolysis process.Biotechnoiogy and Bioengineering,1995,45(4):366-373.
[40]Melander.M.Determination of the degree of polymerization of carboxymethyl cellulose by size exclusion chromatography.Carbohydrate Polymers.2001,46(3):227-233.
[41]张元琴,黄勇.以纤维素材料为基质的生物降解材料的研究进展.高分子材料科学与工程,1999,15(5):25-29.
[42]Ramos L.P.Nazhad M.and Saddler J.N.Cffect of enzymatic hydrolysis on the morphology and fine structure of pretreated cellulosic residues.Enzyme Microb.Technol,1993.15:821-831.
[43]刘小杰,何国庆,陈启和.康宁木霉ZJ5纤维素发酵培养基的优化[J].浙江大学学报(工学版),2003,37(5):623-628.
[44]Gao P J,Qu Y B,Zhao X,et al.Screening microbial strain for improving the nutritional value of wheat and corn straws as animal feed.Enzyme Microb Technol,1997,20:581-584.
[45]郑亚平,余晓斌.碳源对绿色木霉ZC产中性纤维素酶的影响[J].无锡轻工大学学报,2003,22(2):30-33.
[46]宋国安.纤维素酶的应用开发前景[J].河南化工,1999,6(5):32.
[47]扬雪霞,陈洪章,李佐虎.添加纤维素酶的青贮研究进展[J].生物技术通报,2001(1):37-38.
[48]王建兵,范石军,刘静波等.细菌接种剂和酶制剂在秸秆类饲料青贮中的应用[J].饲料博览,1999,11(4):17-19.
[49]周德宝.青贮饲料的研究、发展及现状[J].氨基酸和生物资源,2004,(2):32-34.
[50]Wilkinson J M,Stark B.Silage in Westem Europe[C].A Survey of 17 Countries.Marlow,UK:Chalcombe Publications,1987.
[51]李培庆,崔艳红,胡海霞.生物技术在提高秸秆饲料利用率上的研究与应用[J].安徽农业科学,2006,34(10):2074-2076.
[52]王富生,马俊孝,任红卫.微生物青贮剂在玉米秸秆青贮中的作用[J].山东大学学报,2004,39(2):112-115.
[53]杨志刚,沈益新,陈阿琴.纤维素酶在青贮饲料的应用[J].饲料博览2002(1):39-41.
[54]丁健,贾亚红,陈小莲等.纤维素酶对玉米青贮饲料糖和有机酸生成量的影响[J].饲料工业,2002,23(9):10-11.
[55]王安,张淑芳,钟一民.纤维素复合酶作为青贮饲料添加剂的研究[J].东北农业大学学报.1997,28(4):358-365.
[56]史占全,刘建新,张箐桦.添加酶制剂对青贮玉米秸发酵品质和化学成分的影响[J].浙江农业大学学报,1998,24(6):653-657.
[57]冯怀蓉,龙涛,崔卫东等.利用青贮复合菌剂制作青贮饲料的效果测定[J].新疆农业科学,2006,43(1):75-77.
[58]李新胜,张春喜,孙哲等.加酶高粱青贮对奶牛产奶量的影响[J].中国奶牛,2001(1):26-27.
[59]顾锡慧,杨翔华,马学良.青贮添加剂研究进展[J].辽宁石油化工大学学报,2004,24(3):29-30.
[60]杨新刚,沈益新.纤维素酶制剂在青贮饲料中的应用[J].畜牧与兽医,2002(9):37-39.
[61]杨连玉,众岛芳也.化学和生物处理对玉米秸秆营养价值的影响[J].吉林农业大学学报,2001(1):83-87.
[62]常巧玲,孙建议等.纤维素酶对奶牛营养的研究进展[J].中国饲料,2005(1):18-20.
[63]McAllan A B,Jacobs J L,Merry R J.Factors influencing the amount and pattemof silage effluent production[C].Forage Conservation towards 2000 Braunschweig,1991,368-370.
[64]WANG Z Y,ZHANG J D,Li Y.Liquid medium on cordyceps militaris[J].Joumal of Liaoning of petroleum & chemical technology,2004,24(1):133-138.
[65]张海,左玉萍,利用酶制剂作为青贮饲料添加剂[J].草与畜,1996(1):30-31.
[66]郭金双,赵光永,冯仰廉等.纤维素酶对大麦青贮品质及中型洗涤纤维瘤胃降解率的影响[J].饲料工业,2005,26(3):13-14.
[67]王国仓,李增辉,范秀兰.微生物在青贮饲料中的作用[J].内蒙古畜牧科学,2003(3):55-56.
[68]蔡义民,熊井清雄,廖芷.乳酸菌制剂对青贮饲料发酵品质的改善效果[J].中国农业科学,1995,28,(2):73-82.
[69]国春艳,杜红芳,刁其玉等.乳酸菌的生理功能及在畜牧业中的应用[J].饲料工业,2006,27(2):55-57.
[70]周淑芹,孙文志.酵母培养物[J].中国饲料,2003(5):31-32.
[71]杨富裕,张蕴薇.尿素在青贮料中的应用[J].中国奶牛,2005(5):23-24.
[72]郭松林.纤维素酶及其在畜禽生产中的应用[J].中国饲料,2001,9:23-24.
[73]陈侠甫.应用木10纤维素酶治疗马牛胃肠病[J].中国兽医杂志,1988,(2):32-33.
[74]王贵权等.纤维素酶治疗牛消化道疾病150例.黑龙江畜牧兽医.1991,7:31.
[75]只兴有.纤维素酶治疗奶牛低酸度酒精阳性乳[J].黑龙江畜牧兽医,1991,6:33.
[76]周正红,高孔荣等.纤维素酶在食品发酵工业中的应用及前景.暨南大学学报(自科与医学版).1998,19(5):125-130.
[77]邱立友,朱德育.黑曲霉酶学特性及其在洒精生产中的应用.河南农业大学学报,1993,27(3):91-295.
[78]汪多仁.纤维素酶在纺织工业中的应用[J].四川化工与腐蚀控制,2000,5(3):52-53.
[79]洪洞,黄秀梨.纤维素酶的应用[J].生物学通报,1997,32(12):19.
[80]叶姜瑜.一种纤维素分解鉴别培养基[J].微生物学通报,1997,24(4):251-252.
[81]沈雪亮,夏黎明.产纤维素酶细菌的筛选及酶学特性研究[J].林产化学与工业,2002,22(1);47-48.
[82]张虹等.纤维素酶产生菌的分离和鉴定[J].齐齐哈尔师范学院学报(自然科学 版),1997,17(4):62.
[83]中国科学院微生物研究所细菌分类组.一般细菌常用鉴定方法[M].北京:科学出版社,1978.
[84]东方珠,蔡妙英等编著.常见细菌系统鉴定手册[M].北京:科学出版社,2001.
[85]钱嘉渊译.酶的测定方法.北京:中国轻工业出版社,1992.
[86]中国科学院上海植物生理研究所.二株高活力纤素分解菌N2-78和EA3-867的获得及其特性的比较[J].微生物学报,1978,18(1):27-38.
[87]王淑军等.纤维素酶酶解稻壳条件试验[J].微生物学报,1995,6:354-357.
[88]WANG ZY,ZHANG J D,Li Y.Liquid medium on cordyceps militaris[J].Journal of Liaoning o f petroleum & chemical technology,2004,24(1):133-138.
[89]R.E.布坎南,N.E.吉本斯等编著.伯杰细菌鉴定手册(第八版)[M].北京:科学出版社,1984.