黑曲霉1504产葡萄糖氧化酶的ARTP诱变、产酶条件优化及改良面粉品质研究
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摘要
用新型诱变方法——常温、常压等离子体诱变(ARTP)对产葡萄糖氧化酶(GOD)菌株黑曲霉1504进行诱变育种,并通过响应面分析法优化产酶条件,将所得GOD用于面粉品质改良。通过ARTP诱变,获得酶活力提高较大的正突变菌株12株,其中2株菌株A117、A158的产酶活力提至原始菌株的3.17倍和3.31倍,分别达到172.72 U/mL和180.74 U/mL。通过响应面分析得到优化的发酵条件:葡萄糖110.28 g/L、牛肉蛋白胨5 g/L、硝酸钠5 g/L、碳酸钙37.89 g/L、KCl 0.5 g/L、KH2PO42 g/L、MgSO4·7H2O 0.7 g/L, pH值自然。接种量为0.05(V/V,3×106个孢子/mL),30℃下培养4 d,产酶活力达到373.24 U/mL,是优化前的2.02倍,属国内较高的胞外葡萄糖氧化酶生产水平。GOD对面粉品质改良的研究表明,适量添加GOD的面粉吸水率、形成时间、稳定时间及粉质指数都有所增加,面团拉伸阻力明显增大,延伸度有所下降,说明面粉性质得到改良。
A new mutation method of room temperature and atmospheric pressure plasma mutagenesis(ARTP) was used to improve the production of glucose oxidase(GOD) from Aspergillus niger strains 1504. The optimization of fermentation conditions by response surface analysis and the effect of GOD on the quality of flour were investigated. 12 strains of positive mutant strain were obtained by ARTP mutation. The GOD activity of the mutated strain A117 and A158 reached 172.72 U/mL and 180.74 U/mL, respectively, which were 3.17 and 3.31 times of the original strain. By response surface analysis, optimization of fermentation conditions for GOD was as follows: 0.7 g/L of glucose 110.28 g/L and beef peptone 5 g/L sodium nitrate and 5 g/L, calcium carbonate 37.89 g/L, KCl 0.5 g/L, KH2 PO42 g/L, MgSO47 H2 O, natural pH, inoculation amount is 0.05(V/V, 3 × 106 spores/mL). Under the optimal conditions cultured for 4 d at 30 ℃, the enzyme activity reached 373.24 U/mL which belongs to the domestic high extracellular GOD production level. God flour quality improvement study found that adding God flour water absorption rate, formation time, stability time and Farinograph index increased, significantly increases the tensile resistance of dough. The degree of extensibility decreased, indicating that the flour properties had been improved.
A new mutation method of room temperature and atmospheric pressure plasma mutagenesis(ARTP) was used to improve the production of glucose oxidase(GOD) from Aspergillus niger strains 1504. The optimization of fermentation conditions by response surface analysis and the effect of GOD on the quality of flour were investigated. 12 strains of positive mutant strain were obtained by ARTP mutation. The GOD activity of the mutated strain A117 and A158 reached 172.72 U/mL and 180.74 U/mL, respectively, which were 3.17 and 3.31 times of the original strain. By response surface analysis, optimization of fermentation conditions for GOD was as follows: 0.7 g/L of glucose 110.28 g/L and beef peptone 5 g/L sodium nitrate and 5 g/L, calcium carbonate 37.89 g/L, KCl 0.5 g/L, KH2 PO42 g/L, MgSO47 H2 O, natural pH, inoculation amount is 0.05(V/V, 3 × 106 spores/mL). Under the optimal conditions cultured for 4 d at 30 ℃, the enzyme activity reached 373.24 U/mL which belongs to the domestic high extracellular GOD production level. God flour quality improvement study found that adding God flour water absorption rate, formation time, stability time and Farinograph index increased, significantly increases the tensile resistance of dough. The degree of extensibility decreased, indicating that the flour properties had been improved.
引文
[1]朱运平,伍少明,李秀婷,等.微生物葡萄糖氧化酶的生产及其在食品工业中应用的研究进展[J].中国食品添加剂, 2013,(5):165-172.
[2]韩建春,冯镇,张宏伟.产葡萄糖氧化酶菌株的筛选及发酵培养基的优化[J].食品科学, 2011, 32(1)149-153.
[3]赵秀梅.葡聚糖酶、葡萄糖氧化酶对低筋粉馒头的品质改良[J].中国食品添加剂, 2004,(3):83-85.
[4]李艳,李静.葡萄糖氧化酶及其在酒中的应用[J].酿酒, 2006, 33(5):63-66.
[5]范新蕾.产葡萄糖氧化酶菌株的诱变选育及发酵条件优化[D].无锡:江南大学, 2014.
[6] DE OLIVEIRA A C A, ASSIS V C, MATOS M A C, et al. Flow-injection system with glucose oxidase immobilizedon a tubular reactor for determination of glucose in blood samples[J]. Analytica Chimica Acta,2005, 535(1):213-217.
[7] ZHANG W, HUANG Y, DAI H, et al. Tuning the redox and enzymatic activity of glucose oxidase in layered organic films and its application in glucose biosensors[J]. Analytical Biochemistry, 2004, 329(1):85-90
[8]方军成,尤瑞祺,宋聪,等.葡萄糖氧化酶的生产及其在饲料工业中的应用[J].饲料博览, 2015(10):1-7.
[9]蔡友华,李文锋,卢伟宁,等.新型常压室温等离子体(ARTP)快速诱变高产苏氨酸的突变株[J].现代食品科技, 2013, 29(8):1888-1892.
[10]万青,曹伟佳,张常青,等.常压室温等离子体诱变高效利用木糖产丁二酸菌株[J].生物工程学报,2013, 29(11):1692-1695.
[11]金丽华,方明月,张翀,等.常压室温等离子体快速诱变产油酵母的条件及其突变株的特性[J].生物工程学报, 2011, 27(3):461-467.
[12] FIEDUREK J, SZCZODRAK J. Glucose oxidase biosynthesis in relation to biochemical mutations in Aspergillus niger[J]. Acta Biotechnologica, 1995, 15(1):107-115.
[13] FIEDUREK J, GROMADA A. Screening and mutagenesis of molds for improvement of the simultaneous production of catalase and glucose oxidase[J].Enzyme and Microbial Technology, 1997, 20(5):344-347.
[14]朱运平,褚文丹,李秀婷,等. 1株产胞外葡萄糖氧化酶黑曲霉的液体发酵条件优化[J].中国食品学报,2014,14(5):90-97.
[15] PRIYA R, KANMANI S. Optimization of photocatalytic production of hydrogen from hydrogen sulfide in alkaline solution using response surface methodology[J]. Desalination, 2011, 276(1):222-227.
[16]陈韵亿,金丽华,张翀,等.常压室温等离子体非转基因生物诱变木糖代谢产油酵母的方法及特性研究[J].食品科学, 2012, 33(增刊1):212-217.
[17]宫艳艳,徐学明.葡萄糖氧化酶的发酵生产与发酵工艺的研究[J].农业工程技术(农产品加工), 2007(8):18-22.
[18]江洁,刘德宗,王鑫,等.葡萄糖氧化酶发酵工艺的研究[J].齐齐哈尔轻工学院学报, 1996, 12(4):49-52.
[19]梁静娟,李筱瑜,官威,等.产葡萄糖氧化酶黑曲霉的诱变选育及葡萄糖酸钙发酵条件的研究[J].食品工业科技, 2010,(12):218-220.
[20] HARDEEP S G, CRISTINA M. R. Improvement of the breadmaking quality of rice flour by glucose oxidase[J]. Food Research International, 2004, 37:75-81.
[2]韩建春,冯镇,张宏伟.产葡萄糖氧化酶菌株的筛选及发酵培养基的优化[J].食品科学, 2011, 32(1)149-153.
[3]赵秀梅.葡聚糖酶、葡萄糖氧化酶对低筋粉馒头的品质改良[J].中国食品添加剂, 2004,(3):83-85.
[4]李艳,李静.葡萄糖氧化酶及其在酒中的应用[J].酿酒, 2006, 33(5):63-66.
[5]范新蕾.产葡萄糖氧化酶菌株的诱变选育及发酵条件优化[D].无锡:江南大学, 2014.
[6] DE OLIVEIRA A C A, ASSIS V C, MATOS M A C, et al. Flow-injection system with glucose oxidase immobilizedon a tubular reactor for determination of glucose in blood samples[J]. Analytica Chimica Acta,2005, 535(1):213-217.
[7] ZHANG W, HUANG Y, DAI H, et al. Tuning the redox and enzymatic activity of glucose oxidase in layered organic films and its application in glucose biosensors[J]. Analytical Biochemistry, 2004, 329(1):85-90
[8]方军成,尤瑞祺,宋聪,等.葡萄糖氧化酶的生产及其在饲料工业中的应用[J].饲料博览, 2015(10):1-7.
[9]蔡友华,李文锋,卢伟宁,等.新型常压室温等离子体(ARTP)快速诱变高产苏氨酸的突变株[J].现代食品科技, 2013, 29(8):1888-1892.
[10]万青,曹伟佳,张常青,等.常压室温等离子体诱变高效利用木糖产丁二酸菌株[J].生物工程学报,2013, 29(11):1692-1695.
[11]金丽华,方明月,张翀,等.常压室温等离子体快速诱变产油酵母的条件及其突变株的特性[J].生物工程学报, 2011, 27(3):461-467.
[12] FIEDUREK J, SZCZODRAK J. Glucose oxidase biosynthesis in relation to biochemical mutations in Aspergillus niger[J]. Acta Biotechnologica, 1995, 15(1):107-115.
[13] FIEDUREK J, GROMADA A. Screening and mutagenesis of molds for improvement of the simultaneous production of catalase and glucose oxidase[J].Enzyme and Microbial Technology, 1997, 20(5):344-347.
[14]朱运平,褚文丹,李秀婷,等. 1株产胞外葡萄糖氧化酶黑曲霉的液体发酵条件优化[J].中国食品学报,2014,14(5):90-97.
[15] PRIYA R, KANMANI S. Optimization of photocatalytic production of hydrogen from hydrogen sulfide in alkaline solution using response surface methodology[J]. Desalination, 2011, 276(1):222-227.
[16]陈韵亿,金丽华,张翀,等.常压室温等离子体非转基因生物诱变木糖代谢产油酵母的方法及特性研究[J].食品科学, 2012, 33(增刊1):212-217.
[17]宫艳艳,徐学明.葡萄糖氧化酶的发酵生产与发酵工艺的研究[J].农业工程技术(农产品加工), 2007(8):18-22.
[18]江洁,刘德宗,王鑫,等.葡萄糖氧化酶发酵工艺的研究[J].齐齐哈尔轻工学院学报, 1996, 12(4):49-52.
[19]梁静娟,李筱瑜,官威,等.产葡萄糖氧化酶黑曲霉的诱变选育及葡萄糖酸钙发酵条件的研究[J].食品工业科技, 2010,(12):218-220.
[20] HARDEEP S G, CRISTINA M. R. Improvement of the breadmaking quality of rice flour by glucose oxidase[J]. Food Research International, 2004, 37:75-81.