高温乳糖酶高产菌株发酵条件优化及酶制剂的研究
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摘要
乳糖酶是一种水解酶,目前主要应用于食品、医药、分析等领域。本论文以本实验室保藏的高产乳糖酶菌株Aspergillus niger DL116及其发酵的乳糖酶为研究对象,采用已优化的发酵培养基进行了发酵条件优化及10L发酵罐的放大试验,并对固体及液体乳糖酶酶制剂的制备进行了初步研究,为其工业化生产打下了基础。黑曲霉DL116乳糖酶是胞外高温乳糖酶,具有生产成本低、稳定性好、在生产中可以有效防止杂菌污染等特点,具有广阔的应用前景。
在对黑曲霉DL116的摇瓶发酵条件优化的过程中,采用菌丝体方式接种,分别考察了接种量、温度、起始pH值、溶氧对发酵产酶的影响。通过单因素试验和正交试验确定了产乳糖酶的最佳发酵条件为:摇床28℃,200rpm,起始pH为6.0,接种量为6%,装液量为20mL/300mL。经发酵条件优化后,乳糖酶的活力达到116.54U/mL,比未优化前提高了22.85%,且发酵周期缩短了2d。
以摇瓶发酵试验为基础,进行了10L发酵罐的放大试验,测定了包括相对溶氧值、pH值、总糖、还原糖、氨基氮、乳糖酶活性在内的代谢情况,为发酵放大奠定了基础。
研究了不同物质对乳糖酶热稳定性的影响,结果表明添加甘油、乳糖、麦芽糊精、大豆蛋白等可提高乳糖酶的热稳定性,结合单因素试验,采用正交实验设计优化出了最佳复合稳定剂为4%甘油、3%乳糖、4%麦芽糊精、4%大豆蛋白,在该稳定剂作用下,经70℃保温2h后,酶活保留率仍为85.28%,可显著提高乳糖酶的热稳定性。
发酵液经过滤、超滤得到浓缩酶液;向浓缩酶液中添加上述复合稳定剂和0.1%防腐剂的液体酶制剂,在室温条件下储存60d,酶活保留率为88.77%,比对照高20.63%,显示出良好的储存稳定性。该液体酶制剂的酶活力为424.84U/mL,最适作用温度为70℃,最适作用pH4.0。
按照酶液:乙醇(V/V)为1:2.5的比例向浓缩酶液中添加乙醇进行沉淀后,采用真空干燥制得固体酶制剂。该固体酶制剂的酶活力为14286.19U/g,最适作用温度为70℃,最适作用pH4.0,在室温条件下储存30d,酶活保留率为96.46%。
本研究建立了黑曲霉菌株DL116产乳糖酶的摇瓶优化发酵条件和液体乳糖酶制剂的复合稳定剂,并研制出了乳糖酶液体酶制剂和固体酶制剂,为最终乳糖酶的工业化生产摸索和创造条件,改善一直以来我国乳糖酶依赖进口的状况。
Lactase is a kind of hydrolase that mainly applied in fields such as food, medicine, analyzing, etc. at present. Optimization of fermentation conditions in shaking fask level of producing lactase and fermentation in 10L fermentor are investigsted by the Aspergillus niger DL116 which was stored in our lab in this thesis. The preparation processing of solid and liquid enzyme was studied primarily, the enzyme properties which will be a foundation for its industrial production. The Aspergillus niger DL116 lactase which has a broad application prospects because of its low production costs, fine stability, effective prevention on other strains in the production is a extracelluar and high temperature enzyme. The main results were as following:
On the process of optimizing fermentation condition, impact of inoculation amount, temperature, initial pH, dissolution oxygen on fermentation by mycelium inoculation have been studied in shake flask level. The fermentation conditions was optimized by single-factor and orthogonal design experiments. The optimum fermentation conditions on producing lactase were as follows: 28°C, initial pH6.0, inoculation amount 6%, flask liquid volume 20mL/300mL. After optimization of fermentation condition, the lactase activity can reach 116.54U/mL, which increased 22.85% before optimization and the fermentation periods shortened 2 days.
Based on the results at shake flask level, scale up studies were carried out in 10L fermentor. The metabolizing parameters including dissolution oxygen, pH, total sugar, deoxidize sugar, ammonia nitrogen and lactase activity were tested. It laid a foundation for large scale fermentation.
In order to increase the lactase thermal stability, the effects of different substances were investigated. The results showed that the thermal stability of lactase could be increased for adding glycerol, lactose, maltodextrin, soy protein etc. into the lactase solution. Based on the results of single factor experiments, an orthogonal experiment design was used to optimize the best compounding of combined protective agent which including 4% glycerol, 3% lactose, 4% maltodextrin, 4% soy protein. Under the protection of the combined protective agent, the enzyme activity remained 85.28% after incubation of 2h at 70°C. So the combined protective agent had a significant effect on the enhancement of the lactase thermal stability.
The concentrated enzyme liquid was gained by filtration and ultrafiltraion. The combined protective agent and 0.1% sodium benzoate were added to the concentrated enzyme liquid, the liquid lactase preparation showed good storage stability, The lactase activity was 88.77% stored on room temperature for 60 days. It was increased by 20.63% comparing with the control. The lactase activity is 424.84U/mL of the liquid lactase preparation, the optimum temperature is 70°C and pH is 4.0.
After ultrafiltraion, the condensated lactase was been sedimentated by ethanol. Finally the solid enzyme was gotten by vacuum drying. The lactase activity is 14286.19U/g of the solid lactase preparation, the optimum temperature is 70°C and pH is 4.0. The lactase activity was 96.46% stored on room temperature for 30 da>s.
在对黑曲霉DL116的摇瓶发酵条件优化的过程中,采用菌丝体方式接种,分别考察了接种量、温度、起始pH值、溶氧对发酵产酶的影响。通过单因素试验和正交试验确定了产乳糖酶的最佳发酵条件为:摇床28℃,200rpm,起始pH为6.0,接种量为6%,装液量为20mL/300mL。经发酵条件优化后,乳糖酶的活力达到116.54U/mL,比未优化前提高了22.85%,且发酵周期缩短了2d。
以摇瓶发酵试验为基础,进行了10L发酵罐的放大试验,测定了包括相对溶氧值、pH值、总糖、还原糖、氨基氮、乳糖酶活性在内的代谢情况,为发酵放大奠定了基础。
研究了不同物质对乳糖酶热稳定性的影响,结果表明添加甘油、乳糖、麦芽糊精、大豆蛋白等可提高乳糖酶的热稳定性,结合单因素试验,采用正交实验设计优化出了最佳复合稳定剂为4%甘油、3%乳糖、4%麦芽糊精、4%大豆蛋白,在该稳定剂作用下,经70℃保温2h后,酶活保留率仍为85.28%,可显著提高乳糖酶的热稳定性。
发酵液经过滤、超滤得到浓缩酶液;向浓缩酶液中添加上述复合稳定剂和0.1%防腐剂的液体酶制剂,在室温条件下储存60d,酶活保留率为88.77%,比对照高20.63%,显示出良好的储存稳定性。该液体酶制剂的酶活力为424.84U/mL,最适作用温度为70℃,最适作用pH4.0。
按照酶液:乙醇(V/V)为1:2.5的比例向浓缩酶液中添加乙醇进行沉淀后,采用真空干燥制得固体酶制剂。该固体酶制剂的酶活力为14286.19U/g,最适作用温度为70℃,最适作用pH4.0,在室温条件下储存30d,酶活保留率为96.46%。
本研究建立了黑曲霉菌株DL116产乳糖酶的摇瓶优化发酵条件和液体乳糖酶制剂的复合稳定剂,并研制出了乳糖酶液体酶制剂和固体酶制剂,为最终乳糖酶的工业化生产摸索和创造条件,改善一直以来我国乳糖酶依赖进口的状况。
Lactase is a kind of hydrolase that mainly applied in fields such as food, medicine, analyzing, etc. at present. Optimization of fermentation conditions in shaking fask level of producing lactase and fermentation in 10L fermentor are investigsted by the Aspergillus niger DL116 which was stored in our lab in this thesis. The preparation processing of solid and liquid enzyme was studied primarily, the enzyme properties which will be a foundation for its industrial production. The Aspergillus niger DL116 lactase which has a broad application prospects because of its low production costs, fine stability, effective prevention on other strains in the production is a extracelluar and high temperature enzyme. The main results were as following:
On the process of optimizing fermentation condition, impact of inoculation amount, temperature, initial pH, dissolution oxygen on fermentation by mycelium inoculation have been studied in shake flask level. The fermentation conditions was optimized by single-factor and orthogonal design experiments. The optimum fermentation conditions on producing lactase were as follows: 28°C, initial pH6.0, inoculation amount 6%, flask liquid volume 20mL/300mL. After optimization of fermentation condition, the lactase activity can reach 116.54U/mL, which increased 22.85% before optimization and the fermentation periods shortened 2 days.
Based on the results at shake flask level, scale up studies were carried out in 10L fermentor. The metabolizing parameters including dissolution oxygen, pH, total sugar, deoxidize sugar, ammonia nitrogen and lactase activity were tested. It laid a foundation for large scale fermentation.
In order to increase the lactase thermal stability, the effects of different substances were investigated. The results showed that the thermal stability of lactase could be increased for adding glycerol, lactose, maltodextrin, soy protein etc. into the lactase solution. Based on the results of single factor experiments, an orthogonal experiment design was used to optimize the best compounding of combined protective agent which including 4% glycerol, 3% lactose, 4% maltodextrin, 4% soy protein. Under the protection of the combined protective agent, the enzyme activity remained 85.28% after incubation of 2h at 70°C. So the combined protective agent had a significant effect on the enhancement of the lactase thermal stability.
The concentrated enzyme liquid was gained by filtration and ultrafiltraion. The combined protective agent and 0.1% sodium benzoate were added to the concentrated enzyme liquid, the liquid lactase preparation showed good storage stability, The lactase activity was 88.77% stored on room temperature for 60 days. It was increased by 20.63% comparing with the control. The lactase activity is 424.84U/mL of the liquid lactase preparation, the optimum temperature is 70°C and pH is 4.0.
After ultrafiltraion, the condensated lactase was been sedimentated by ethanol. Finally the solid enzyme was gotten by vacuum drying. The lactase activity is 14286.19U/g of the solid lactase preparation, the optimum temperature is 70°C and pH is 4.0. The lactase activity was 96.46% stored on room temperature for 30 da>s.
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