白腐真菌生物降解油菜秸秆及饲料化研究
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摘要
本试验选择以产漆酶为主的野生菌糙皮侧耳(Pleurotus ostreatusWY-01)为研究材料,利用常规的紫外线照射技术对其进行改良育种,筛选出一株高产菌株ADW-08,然后对其固体发酵培养基、最佳发酵条件进行了初步优化,并对ADW-08木质素降解酶系组成及其对油菜秸秆生物降解效果进行了研究,同时对油菜秸秆饲料化应用作了有益探索。
对菌糙皮侧耳(Pleurotus ostreatusWY-01)进行紫外线照射,结果表明:当照射时间为50s时,其孢子致死率在70%～80%之间,而此时突变率达到最大,因此确定50s为最佳照射时间。突变高产菌株ADW-08的漆酶活性由出发菌株PleurotusostreatusWY-01的最高酶活力从2.78 U/g提高到7.78U/g。突变菌株ADW-08经传5代培养,产酶性能稳定。
采用单因素试验和正交试验对ADW-08产漆酶固体培养基进行初步研究,确定其最适培养基组成:葡萄糖15g/L,酒石酸铵0.4g/L,硫酸锰0.25g/L。在优化后的培养基基础上确定其产漆酶最适发酵条件为:发酵温度为28℃、培养基起始pH为5.0、接种量为每20g油菜秸秆3个菌塞(φ12cm)时漆酶分泌量最大。ABTS(1mmol/L)和藜芦醇(1mmol/L)对漆酶的分泌均有不同程度的诱导作用,而吐温-80(1%)对产漆酶有一定的抑制作用。
高产菌株ADW-08的木质素降解率最佳固体配方组成:葡萄糖15g/L,酒石酸铵0.4g/L,硫酸锰0.75g/L。
对高产菌株ADW-08木质素降解酶系组成和生物降解油菜秸秆效果研究表明:高产菌株ADW-08木质素降解酶酶系为Laccase-MnP型,其最高酶活分别出现在第24天6.67U/g和2.67U/g;发酵60天时,油菜秸秆中木质素、纤维素和半纤维素降解率分别为56.28%、40.80%和45.22%,粗蛋白、必需氨基酸和总氨基酸分别提高43.73%、135.16%和101.51%,且氨基酸含量齐全。
油菜菌糠作为饲料部分替代奶牛日粮进行饲喂试验结果表明:替代50%苜蓿干草饲喂泌乳牛对奶牛生产性能、乳品质和血清生化指标无不良影响,能明显降低奶牛饲养成本,提高奶牛养殖经济效益。
The strain Pleurotus ostreatus WY01 was chose as studying material in this experiment.Firstly,the strain was mutagenesised by the ultraviolet irradiation,and a high producing strain ADW-08 was screened.Then,the culture medium and the fermentation conditions of the mutagenic strain ADW-08 were studied.Finally,the composition of ligninolytic enzyme excreted by ADW-08,the rape straw biodegradation by ADW-08 and application for its fermentation residue for cows were studied.
The ultraviolet irradiation indicated:As the irradiated time was 50 seconds,the lethality rate of spores was between 70%and 80%and the mutation rate was at the highest level,so 50s was definited as the optimum irradiated time.The peak activity of Laccase of mutated strain ADW-08 was raised from 2.78U/g excreted by Pleurotus ostreatus WY01 to 7.78U/g.Characteristics of enzymes production in ADW-08 kept stable by 5 times subculture.
By single and orthogonal test,a culture medium for the Laccase production by ADW-08 was studied.The optimal solid medium consists of 15g/L glucose,0.4g/L ammonium tartrate,0.25g/L MnSO_4·H_2O.The study on optimal fermentation conditions of the mutagenic strain ADW-08 was indicated:the optimum fermentation temperature was 28℃,pH was 5.0 and seed volume was 3 germ bungs/20g.Laccase was induced effectively by ABTS(1mmol/L) and veratryl alcohol(1mmol/L),and inhibited by Tween-80(1%).
The optimal solid medium for lignin biodegradation consists of 15g/L glucose,0.4g/L ammonium tartrate,0.75g/L MnSO_4·H_2O.
The study on the ligninolytic enzyme composition of ADW-08 and the rape straw biodegradation indicated:the ligninolytic enzyme composition of ADW-08 were Laccase and MnP.The peak values of Lacease and MnP were 6.67 U/g and 2.67U/g at 24th day respectively.By 60 days fermentation,the degradation rates of lignin,cellulose and hemicellulose were 56.28%,40.80%and 45.22%respectively,and the content of crude protein,essential amino acid and total amino acid were raised by 43.73%,135.17%and 101.51%,respectively.
The study on cow indicated:there was no harmful influence by 50%alfalfa hays replaced by rape substrates in cows' diet on production performance,milk quality and serum biological parameters.The cost was obviously cut down and the economic returns was largely raised.
对菌糙皮侧耳(Pleurotus ostreatusWY-01)进行紫外线照射,结果表明:当照射时间为50s时,其孢子致死率在70%～80%之间,而此时突变率达到最大,因此确定50s为最佳照射时间。突变高产菌株ADW-08的漆酶活性由出发菌株PleurotusostreatusWY-01的最高酶活力从2.78 U/g提高到7.78U/g。突变菌株ADW-08经传5代培养,产酶性能稳定。
采用单因素试验和正交试验对ADW-08产漆酶固体培养基进行初步研究,确定其最适培养基组成:葡萄糖15g/L,酒石酸铵0.4g/L,硫酸锰0.25g/L。在优化后的培养基基础上确定其产漆酶最适发酵条件为:发酵温度为28℃、培养基起始pH为5.0、接种量为每20g油菜秸秆3个菌塞(φ12cm)时漆酶分泌量最大。ABTS(1mmol/L)和藜芦醇(1mmol/L)对漆酶的分泌均有不同程度的诱导作用,而吐温-80(1%)对产漆酶有一定的抑制作用。
高产菌株ADW-08的木质素降解率最佳固体配方组成:葡萄糖15g/L,酒石酸铵0.4g/L,硫酸锰0.75g/L。
对高产菌株ADW-08木质素降解酶系组成和生物降解油菜秸秆效果研究表明:高产菌株ADW-08木质素降解酶酶系为Laccase-MnP型,其最高酶活分别出现在第24天6.67U/g和2.67U/g;发酵60天时,油菜秸秆中木质素、纤维素和半纤维素降解率分别为56.28%、40.80%和45.22%,粗蛋白、必需氨基酸和总氨基酸分别提高43.73%、135.16%和101.51%,且氨基酸含量齐全。
油菜菌糠作为饲料部分替代奶牛日粮进行饲喂试验结果表明:替代50%苜蓿干草饲喂泌乳牛对奶牛生产性能、乳品质和血清生化指标无不良影响,能明显降低奶牛饲养成本,提高奶牛养殖经济效益。
The strain Pleurotus ostreatus WY01 was chose as studying material in this experiment.Firstly,the strain was mutagenesised by the ultraviolet irradiation,and a high producing strain ADW-08 was screened.Then,the culture medium and the fermentation conditions of the mutagenic strain ADW-08 were studied.Finally,the composition of ligninolytic enzyme excreted by ADW-08,the rape straw biodegradation by ADW-08 and application for its fermentation residue for cows were studied.
The ultraviolet irradiation indicated:As the irradiated time was 50 seconds,the lethality rate of spores was between 70%and 80%and the mutation rate was at the highest level,so 50s was definited as the optimum irradiated time.The peak activity of Laccase of mutated strain ADW-08 was raised from 2.78U/g excreted by Pleurotus ostreatus WY01 to 7.78U/g.Characteristics of enzymes production in ADW-08 kept stable by 5 times subculture.
By single and orthogonal test,a culture medium for the Laccase production by ADW-08 was studied.The optimal solid medium consists of 15g/L glucose,0.4g/L ammonium tartrate,0.25g/L MnSO_4·H_2O.The study on optimal fermentation conditions of the mutagenic strain ADW-08 was indicated:the optimum fermentation temperature was 28℃,pH was 5.0 and seed volume was 3 germ bungs/20g.Laccase was induced effectively by ABTS(1mmol/L) and veratryl alcohol(1mmol/L),and inhibited by Tween-80(1%).
The optimal solid medium for lignin biodegradation consists of 15g/L glucose,0.4g/L ammonium tartrate,0.75g/L MnSO_4·H_2O.
The study on the ligninolytic enzyme composition of ADW-08 and the rape straw biodegradation indicated:the ligninolytic enzyme composition of ADW-08 were Laccase and MnP.The peak values of Lacease and MnP were 6.67 U/g and 2.67U/g at 24th day respectively.By 60 days fermentation,the degradation rates of lignin,cellulose and hemicellulose were 56.28%,40.80%and 45.22%respectively,and the content of crude protein,essential amino acid and total amino acid were raised by 43.73%,135.17%and 101.51%,respectively.
The study on cow indicated:there was no harmful influence by 50%alfalfa hays replaced by rape substrates in cows' diet on production performance,milk quality and serum biological parameters.The cost was obviously cut down and the economic returns was largely raised.
引文
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