亮斑扁角水虻（Hermetia illucens L.）幼虫肠道内纤维素酶活性菌株的筛选与鉴定
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摘要
猪粪,牛粪中的粗纤维经亮斑扁角水虻幼虫生物转化之后有明显的降低,水虻肠道微生物在其中也起着比较重要的作用。从环境保护和发现利用新的菌种资源角度来讲,有必要对亮斑扁角水虻幼虫肠道内具有纤维素酶活的菌株进行鉴定和分析。
本课题以猪粪饲养的亮斑扁角水虻幼虫为研究对象,在对猪粪消化能力最强的4龄幼虫肠道内筛选并鉴定了七株具有纤维素酶活性的细菌；通过进一步比较分析,获得一株纤维素酶活性较高的菌株X4；分别提取猪粪和水虻处理后的猪粪残渣中的粗纤维,使用X4菌株对其进行降解；设计正交试验,对使用X4菌株生产纤维素酶的发酵条件进行优化,为其应用提供参考。
试验结果：从水虻幼虫肠道内筛选出七株具有纤维素酶活性的菌株X1,X2,X3,X4, X5, X6, X7并做系统进化树分析；抑菌试验表明：X1,X2和X3菌对核盘菌无抑制作用,X4, X5, X6, X7菌对核盘菌有明显的抑制作用,其中X4菌对大肠杆菌有抑制作用,X5对金黄色葡萄球菌有抑制作用。酶活测定结果表明,在相同条件下X4菌株纤维素酶活为7个国际单位。
测定X4菌株的生长曲线时发现,5-7小时可达到对数生长期。提取新鲜猪粪和水虻幼虫转化后的猪粪残渣中粗纤维,使用X4菌株进行降解,培养48小时后计算降解率,发现猪粪中12.64%的干物质被降解,残渣中10%的干物质被降解。
用玉米粉和豆粕粉配制产酶发酵培养基,对X4发酵培养条件进行优化。结果显示,培养基初始pH为9,发酵温度45℃,发酵时间36小时,产酶活性最高,可达117.13 IU,比优化前提高了近17倍。这为工业化生产纤维素酶提供了参考。
The coarse fiber of swine manure and shard was significantly low after digesting by Hermetia illucens L. maggots and during the process microbe in intestines of maggots may play an important role. Considering the environmental protection and utilization new strains, it was necessary to screen and identify strains with enzyme activity of cellulose. Seven strains with the activity of cellulose had been screened and identified from the 4th larvae when they had the best digestion to swine manure. Through the determination of CMCase activity and FPA activity, obtain a strain named X4 with high cellulose activity. Extract the crude fiber from swine manure and residue that had been digested by Hermetia illucens L. maggots and degrade with X4. Design orthogonal test to optimize the conditions of fermentation. The results were as follow.
Seven strains had been screened. There were named X1, X2, X3, X4, X5, X6 and X7. Determine the seven strains by the methods of morphological characteristics and 16s rDNA. The antibiotic spectra of the seven showed X4 could inhibit Escherichia coli and Sclerotinia sclerotiorum, X5 could inhibit Staphylococcus aureus and Sclerotinia sclerotiorum, X6 and X7 could inhibit Sclerotinia sclerotiorum. Determine enzyme activity of the seven strains with the methods of DNS and FPA. X4 had the highest enzyme activity (7 IU).
Determine the X4-growth curve with beef cream-peptone culture medium. The result showed that the logarithmic growth phase of X4 was 5-7h. Culture them for 48h and calculate the degradation rate. The results were that the degradation rate of swine manure was 12.64% and the degradation rate of residue was 10%.
Make the fermentation medium with corn and soybean. The optimal condition of producing cellulose was studied by three factors and three levels orthogonal experiment and the results were the best initial pH 9.0, incubating temperature 45℃and incubating time 36h. The enzyme activity was 117.131U and increased nearly 17 times than before. Introduced reliable technical parameters are suitable for commercial production
本课题以猪粪饲养的亮斑扁角水虻幼虫为研究对象,在对猪粪消化能力最强的4龄幼虫肠道内筛选并鉴定了七株具有纤维素酶活性的细菌；通过进一步比较分析,获得一株纤维素酶活性较高的菌株X4；分别提取猪粪和水虻处理后的猪粪残渣中的粗纤维,使用X4菌株对其进行降解；设计正交试验,对使用X4菌株生产纤维素酶的发酵条件进行优化,为其应用提供参考。
试验结果：从水虻幼虫肠道内筛选出七株具有纤维素酶活性的菌株X1,X2,X3,X4, X5, X6, X7并做系统进化树分析；抑菌试验表明：X1,X2和X3菌对核盘菌无抑制作用,X4, X5, X6, X7菌对核盘菌有明显的抑制作用,其中X4菌对大肠杆菌有抑制作用,X5对金黄色葡萄球菌有抑制作用。酶活测定结果表明,在相同条件下X4菌株纤维素酶活为7个国际单位。
测定X4菌株的生长曲线时发现,5-7小时可达到对数生长期。提取新鲜猪粪和水虻幼虫转化后的猪粪残渣中粗纤维,使用X4菌株进行降解,培养48小时后计算降解率,发现猪粪中12.64%的干物质被降解,残渣中10%的干物质被降解。
用玉米粉和豆粕粉配制产酶发酵培养基,对X4发酵培养条件进行优化。结果显示,培养基初始pH为9,发酵温度45℃,发酵时间36小时,产酶活性最高,可达117.13 IU,比优化前提高了近17倍。这为工业化生产纤维素酶提供了参考。
The coarse fiber of swine manure and shard was significantly low after digesting by Hermetia illucens L. maggots and during the process microbe in intestines of maggots may play an important role. Considering the environmental protection and utilization new strains, it was necessary to screen and identify strains with enzyme activity of cellulose. Seven strains with the activity of cellulose had been screened and identified from the 4th larvae when they had the best digestion to swine manure. Through the determination of CMCase activity and FPA activity, obtain a strain named X4 with high cellulose activity. Extract the crude fiber from swine manure and residue that had been digested by Hermetia illucens L. maggots and degrade with X4. Design orthogonal test to optimize the conditions of fermentation. The results were as follow.
Seven strains had been screened. There were named X1, X2, X3, X4, X5, X6 and X7. Determine the seven strains by the methods of morphological characteristics and 16s rDNA. The antibiotic spectra of the seven showed X4 could inhibit Escherichia coli and Sclerotinia sclerotiorum, X5 could inhibit Staphylococcus aureus and Sclerotinia sclerotiorum, X6 and X7 could inhibit Sclerotinia sclerotiorum. Determine enzyme activity of the seven strains with the methods of DNS and FPA. X4 had the highest enzyme activity (7 IU).
Determine the X4-growth curve with beef cream-peptone culture medium. The result showed that the logarithmic growth phase of X4 was 5-7h. Culture them for 48h and calculate the degradation rate. The results were that the degradation rate of swine manure was 12.64% and the degradation rate of residue was 10%.
Make the fermentation medium with corn and soybean. The optimal condition of producing cellulose was studied by three factors and three levels orthogonal experiment and the results were the best initial pH 9.0, incubating temperature 45℃and incubating time 36h. The enzyme activity was 117.131U and increased nearly 17 times than before. Introduced reliable technical parameters are suitable for commercial production
引文
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2.安新城.黑水虻的生物学特性及营养价值.养殖与饲料,2007,11：67-68
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19.梁靖.纤维素酶在速溶茶中的应用研究.茶叶,2002,28：25-26
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21.詹耀才.麦麸加工下脚料提取纤维素、半纤维素的工艺研究.粮食加工,2008,33：28-30
22.戴红霞.稻草秸杆纤维素生产酒精工艺技术条件探索.大庆石油学院,2005
23.刘春芬.纤维素酶及应用现状.粮食与油脂,2004,1：15-17
24.刘树立.纤维素酶分子结构及作用机理的研究进展.食品科技,2007,7：12-15
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27.张金龙.纤维素酶的生产及其在纺织上的应用.广东化工,1999,2：107-108
28.张壮志.高效纤维素分解菌的筛选、鉴定及其生长条件和产酶活性的研究.山东农业大学图书馆,2009
29.杨玉华.纤维素酶提高食醋产量的研究.食品与发酵工业,1999,25：16-22
30.谢占玲.纤维素酶的研究进展.草业科学,2004,21：72-76
31.赵永勋.微生物菌剂对有机废弃物发酵作用的研究.佳木斯大学学报(自然科学版),2001,19：121-125
32.赵荣乐.纤维素酶研究进展.喀什师范学院学报,2005,26：23-25
33.陈晓春.纤维素酶对肉鸡生产性能和营养物质消化利用率的影响.饲料研究,2005,11：7-9
34.马双青.复合纤维素酶对奶牛产奶量及乳脂率的影响.黑龙江畜牧兽医,2008,4：37-38
35.龚兰芳.生物过滤器处理畜禽业中恶臭气体的研究.可再生能源,2009,22：15-17
36. Study on dynamic changes of rumen microflora and cellulase activities on cashmere goats fed by the two different ratios of concentrate to roughage diet,2005, 20:113-116
37. Baoqing, X.. Mechanism of Cellulase and Laccase in Waste Paper De-inking,2007, The International Symposium
38. Chun-ming, G.. Microbial safety control of compost material with cow dung by heat treatment.环境科学学报(英文版),2007,19(8)：127-130
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2.安新城.黑水虻的生物学特性及营养价值.养殖与饲料,2007,11：67-68
3.朱元招.补饲不同配方酶制剂对断奶犊牛生产性能的影响.中国草食动物,2001,3：15-16
4.何刚强,堵国成.从白蚁中分离筛选纤维素分解菌及其产酶性质.食品与生物技术学报,2009,28：352-355
5.巫小琴.纤维素酶产生菌的分离及其酶活力测定.安徽农业科学,2009,37：17323-17325,17327
6.李作美.纤维素酶高产菌株选育的研究进展.安徽农学通报,2009,15：45-47
7.李健.酱油渣中黄酮及纤维素提取工艺的研究.农产品加工学刊,2008,7：200-202
8.李雯纤.维素酶高产菌株的研究进展.酿酒科技,2009,8：105-107
9.沈雪亮.产纤维素酶细菌的筛选及酶学特性研究.林产化学与工业,2002,22：47-51
10.林启训,吴珍泉.水虻幼虫作为鱼饲料的营养评价.饲料研究,1999,5：13-14
11.祁宏伟.不同水平复合纤维素酶对肉牛增重的影响.饲料工业,2000,21：28-29
12.邱定杰.纤维素酶在反刍动物营养中的研究进展.福建省畜牧兽医学会2008年学术年会论文集,2008
13.夏黎明.可再生纤维素资源酶法降解的研究进展.林产化工通讯,1999,33：23-28
14.徐春厚.纤维素酶菌种的鉴定与筛选.中国饲料,2000,13：9-10
15.徐昶.高产纤维素酶菌株的筛选及产酶条件研究.厦门大学学报(自然科学版),2005,44：107-111
16.祝小.枯草芽孢杆菌Pab02产纤维素酶活性的研究.饲料研究,2007,1：61-63
17.祝令香.一株纤维素降解菌的鉴定.农业生物技术学报,2001,9：255-257
18.高春生.纤维素酶在鲤鱼饲料中的应用试验.粮食与饲料工业,2006,9：32-33
19.梁靖.纤维素酶在速溶茶中的应用研究.茶叶,2002,28：25-26
20.葛春辉.两株产纤维素酶芽孢杆菌的筛选及其纤维素酶部分特性.中国土壤学会第十一届全国会员代表大会暨第七届海峡两岸土壤肥料学术交流研讨会论文集,2008
21.詹耀才.麦麸加工下脚料提取纤维素、半纤维素的工艺研究.粮食加工,2008,33：28-30
22.戴红霞.稻草秸杆纤维素生产酒精工艺技术条件探索.大庆石油学院,2005
23.刘春芬.纤维素酶及应用现状.粮食与油脂,2004,1：15-17
24.刘树立.纤维素酶分子结构及作用机理的研究进展.食品科技,2007,7：12-15
25.吕凯,石英尧,高震魁.猪粪的成分及其利用的研究.安徽农业科学,2001,3：373-375
26.孙晓华.纤维素分解菌的分离、筛选及其环境适应性初步研究.科技通报,2005,21：236-241
27.张金龙.纤维素酶的生产及其在纺织上的应用.广东化工,1999,2：107-108
28.张壮志.高效纤维素分解菌的筛选、鉴定及其生长条件和产酶活性的研究.山东农业大学图书馆,2009
29.杨玉华.纤维素酶提高食醋产量的研究.食品与发酵工业,1999,25：16-22
30.谢占玲.纤维素酶的研究进展.草业科学,2004,21：72-76
31.赵永勋.微生物菌剂对有机废弃物发酵作用的研究.佳木斯大学学报(自然科学版),2001,19：121-125
32.赵荣乐.纤维素酶研究进展.喀什师范学院学报,2005,26：23-25
33.陈晓春.纤维素酶对肉鸡生产性能和营养物质消化利用率的影响.饲料研究,2005,11：7-9
34.马双青.复合纤维素酶对奶牛产奶量及乳脂率的影响.黑龙江畜牧兽医,2008,4：37-38
35.龚兰芳.生物过滤器处理畜禽业中恶臭气体的研究.可再生能源,2009,22：15-17
36. Study on dynamic changes of rumen microflora and cellulase activities on cashmere goats fed by the two different ratios of concentrate to roughage diet,2005, 20:113-116
37. Baoqing, X.. Mechanism of Cellulase and Laccase in Waste Paper De-inking,2007, The International Symposium
38. Chun-ming, G.. Microbial safety control of compost material with cow dung by heat treatment.环境科学学报(英文版),2007,19(8)：127-130
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