柠檬苦素降解菌C6降解特性及活性降解酶的分离纯化
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摘要
对从新鲜醋醅中分离到的一株柠檬苦素降解菌C6(Raoultella ornithinolytica)在酸性条件下的柠檬苦素降解特性进行了研究,并对相关酶系进行了分离纯化。分别从菌龄、接种量、培养时间、柠檬苦素浓度4个因素考察了该菌株对柠檬苦素的降解情况,结合响应面优化了该菌株的降解条件,并在最适条件下对其降解速率进行评价。进一步通过硫酸铵沉淀、Sephadex G-100凝胶过滤层析及DEAE-纤维素柱层析对菌株C6发酵上清液中的柠檬苦素降解酶逐步纯化。结果表明,菌龄10 h、底物柠檬苦素质量浓度为4 mg/L、接种量1%的条件下,作用时间为12 h时菌株C6对柠檬苦素的降解速率可达到91.28%±0.17%,其中作用时间9 h以后降解速率显著增加,经纯化后获得相对分子量约为27 ku的电泳纯柠檬苦素降解酶,纯化倍数为9.44。结果可为该菌株及其酶的进一步开发利用提供理论依据。
An strain C6(Raoultella ornithinolytica)with stronger limonin-degrading ability was isolated and obtained from fresh fermented grains of vinegar. Under acidic conditions, degradation conditions of strain C6 were optimized. The effects of the age of strain,the amount of limonin added, the inoculation amount and the culture time on the degradation of limonin were investigated. The limonin-degrading enzyme of strain Raoultella ornithinolytica C6 was purified by Sephadex G-100 gel filtration chromatography and DEAE-cellulose column chromatography with ammonium sulfate precipitation. The results showed that on the basis of single factor, the optimum degradation conditions of strain limonin-degrading were determined by response surface analysis: the age of strain was 10 h, the inoculation of strain was 1%, the dosage of limonin was 4 mg/L, and the culture time was 12 h, under this condition, the degradation rate of strain C6 was 91.28% ±0.17%, and the degradation rate increased rapidly after 9 hours of action. A single band was obtained by SDS-PAGE, and the electrophoretic purity was achieved, and the molecular quality of protease was about 27 ku, the specific activity of protease increased 9.44 times. It provides a theoretical basis for the further development and utilization of this strain and its enzymes.
An strain C6(Raoultella ornithinolytica)with stronger limonin-degrading ability was isolated and obtained from fresh fermented grains of vinegar. Under acidic conditions, degradation conditions of strain C6 were optimized. The effects of the age of strain,the amount of limonin added, the inoculation amount and the culture time on the degradation of limonin were investigated. The limonin-degrading enzyme of strain Raoultella ornithinolytica C6 was purified by Sephadex G-100 gel filtration chromatography and DEAE-cellulose column chromatography with ammonium sulfate precipitation. The results showed that on the basis of single factor, the optimum degradation conditions of strain limonin-degrading were determined by response surface analysis: the age of strain was 10 h, the inoculation of strain was 1%, the dosage of limonin was 4 mg/L, and the culture time was 12 h, under this condition, the degradation rate of strain C6 was 91.28% ±0.17%, and the degradation rate increased rapidly after 9 hours of action. A single band was obtained by SDS-PAGE, and the electrophoretic purity was achieved, and the molecular quality of protease was about 27 ku, the specific activity of protease increased 9.44 times. It provides a theoretical basis for the further development and utilization of this strain and its enzymes.
引文
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[5]孙志高,黄学根,焦必宁.柑橘果实主要苦味成分的分布及橙汁脱苦技术研究[J].食品科学,2005,26(6):146-148.
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[7] ROY A,SARAF S. Limonoids:overview of significant bioactive triterpenes distributed in plants kingdom[J]. Biological and Pharmaceutical Bulletin,2006,29(2):191-201.
[8]张虹,张艳萍.柠檬苦素类似物的研究进展[J].食品与发酵工业,2002,28(2):80-83.
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[10] MAIER V P,BEVERLY G D. Limonin monolactone,the nonbitter precursor responsible for delayed bitterness in certain citrus juices[J]. Journal of Food Science,1968,33(5):488-492.
[11] MAIER V P,HASEGAWA S,HERA E. Limonin D-ring-lactone hydrolase——A new enzyme from citrus seeds[J]. Phytochemistry,1969,8(2):405-407.
[12]刘亮,戚向阳,董绪燕.柑橘中柠檬苦素类似物的研究新进展[J].农产品加工·学刊,2007(7):37-41.
[13]左安连,毛海舫,李琼,等.柑橘类果汁脱苦方法研究综述[J].香料香精化妆品,2008(3):33-39.
[14] LI L J,WU Z Y,YU Y,et al. Development and characterization of anα-l-rhamnosidase mutant with improved thermostability and a higher efficiency for debittering orange juice[J]. Food chemistry,2018,245:1070-1078.
[15]WILSON K W,CRUTCHFIELD C A. Spectrophotometric determination of limonin in orange juice[J]. Journal of Agricultural and Food Chemistry,1968,16(1):118-124.
[16]张菊华,单扬,李高阳.柑橘果汁中的类柠檬苦素及其脱苦方法[J].食品与机械,2005,21(3):72-75.
[17]李睿晓.柚子酒的微生物脱苦技术研究[D].重庆:西南大学,2010:4-8.
[18]王志国.微生物与柑桔汁脱苦[J].食品科学,2000,21(11):10-14.
[19] MARTINEZ-MADRID C,MANJON A,IBORRA J L. Degradation of limonin by entrapped Rhodococcusfascians cells[J]. Biotechnology letters,1989,11(9):653-658.
[20] KITA M,HIRATA Y,MORIGUCHI T,ET A L. Molecular cloning and characterization of a novel gene encoding limonoid UDP glucosyltransferase in Citrus 1[J]. Febs Letters,2000,469(2/3):173-178.
[21] KARIM M R,HASHINAGA F. Preparation and properties of immobilized pummelo limonoid glucosyltransferase[J]. Process Biochemistry,2002,38(5):809-814.
[22]DUA K,KOCHER G S. Enrichment and characterization of limonin degrading microorganisms isolated from kinnow orchard soil,peel and waste site[J]. Applied Biological Research,2017,19(2):187-196.
[23]PURI M,BANERJEE U C. Production,purification and characterization of the debittering enzyme naringinase[J]. Biotechnology Advances,2000,18(3):207-217.
[24]胡阳.蜜柚苦味物的HPLC检测、提取及脱苦菌株的筛选[D].雅安:四川农业大学,2007:36-48.
[25]董晨,张锦华,白宝清,等.柠檬苦素高效降解菌的分离筛选与鉴定[J].食品与发酵工业,2017,43(8):67-72.
[26]纪学芳,师俊玲,张锦华.柠檬苦素降解菌的分离筛选与分类鉴定[J].食品科学,2011,32(15):177-181.
[27]刘青茹,高彦祥.柑桔汁脱苦降酸方法研究进展[J].食品研究与开发,2015,36(13):127-133.
[28]邓丽莉,潘晓倩,生吉平,等.考马斯亮蓝法测定苹果组织微量可溶性蛋白含量的条件优化[J].食品科学,2012,33(24):185-189.
[29]王松林,彭荣,崔榕,等.类柠檬苦素生物转化与脱苦研究进展[J].食品科学,2015,36(9):279-283.
[30] STINCO C M,FERN魣NDEZ-V魣ZQUEZ R,HERNANZ D,et al.Industrial orange juice debittering:Impact on bioactive compounds and nutritional value[J]. Journal of Food Engineering,2013,116(1):155-161.
[31]刘君.柑桔种子中类柠檬苦素提取纯化工艺研究[D].重庆:西南大学,2008:11-13.
[32] HASEGAWA S,PELTON V A,BENNETT R D. Metabolism of limonoids by Arthrobacter globiformis II:basis for a practical means of reducing the limonin content of orange juice by immobilized cells[J]. Journal of Agricultural and Food Chemistry,1983,31(5):1002-1004.
[33] PATIL M B,DHAKE A B. Debittering of citrus fruit juice by naringinase of Penicillium purpurogenum[J]. Int J Engg Res Sci Tech,2014,3:266-270.
[34]高秋芬,倪辉,李利君,等.黑曲霉胞外酶对柑橘果汁的脱苦作用[J].中国食品学报,2014,14(4):156-163.
[35]黄颖颖,陆东和,杨成龙,等.酶法脱苦对琯溪蜜柚汁主要苦味物质及营养风味的影响[J].福建农业学报,2016,31(8):886-891.
[2] SINGH S V I R,JAIN R K,GUPTA A K,et al. Debittering of citrus juices:A review[J]. Journal of Food Science and Technology,2003,40(3):247-253.
[3] HASEGAWA S,HERMAN Z,ORME E D,et al. Biosynthesis of limonoids in citrus:sites and translocation[J]. Phytochemistry,1986,25(12):2783-2785.
[4]蔡护华,桥永文男.柑桔果实中柠檬苦素类化合物的研究现状与展望[J].植物学报,1996,38(4):328-336.
[5]孙志高,黄学根,焦必宁.柑橘果实主要苦味成分的分布及橙汁脱苦技术研究[J].食品科学,2005,26(6):146-148.
[6] GUADAGNI D G,MAIER V P,TURNBAUGH J G. Effect of some citrus juice constituents on taste thresholds for limonin and naringin bitterness[J]. Journal of the Science of Food and Agriculture,1973,24(10):1277-1288.
[7] ROY A,SARAF S. Limonoids:overview of significant bioactive triterpenes distributed in plants kingdom[J]. Biological and Pharmaceutical Bulletin,2006,29(2):191-201.
[8]张虹,张艳萍.柠檬苦素类似物的研究进展[J].食品与发酵工业,2002,28(2):80-83.
[9] HASEGAWA S,BENNETT R D,HERMAN Z,et al. Limonoid glucosides in citrus[J]. Phytochemistry,1989,28(6):1717-1720.
[10] MAIER V P,BEVERLY G D. Limonin monolactone,the nonbitter precursor responsible for delayed bitterness in certain citrus juices[J]. Journal of Food Science,1968,33(5):488-492.
[11] MAIER V P,HASEGAWA S,HERA E. Limonin D-ring-lactone hydrolase——A new enzyme from citrus seeds[J]. Phytochemistry,1969,8(2):405-407.
[12]刘亮,戚向阳,董绪燕.柑橘中柠檬苦素类似物的研究新进展[J].农产品加工·学刊,2007(7):37-41.
[13]左安连,毛海舫,李琼,等.柑橘类果汁脱苦方法研究综述[J].香料香精化妆品,2008(3):33-39.
[14] LI L J,WU Z Y,YU Y,et al. Development and characterization of anα-l-rhamnosidase mutant with improved thermostability and a higher efficiency for debittering orange juice[J]. Food chemistry,2018,245:1070-1078.
[15]WILSON K W,CRUTCHFIELD C A. Spectrophotometric determination of limonin in orange juice[J]. Journal of Agricultural and Food Chemistry,1968,16(1):118-124.
[16]张菊华,单扬,李高阳.柑橘果汁中的类柠檬苦素及其脱苦方法[J].食品与机械,2005,21(3):72-75.
[17]李睿晓.柚子酒的微生物脱苦技术研究[D].重庆:西南大学,2010:4-8.
[18]王志国.微生物与柑桔汁脱苦[J].食品科学,2000,21(11):10-14.
[19] MARTINEZ-MADRID C,MANJON A,IBORRA J L. Degradation of limonin by entrapped Rhodococcusfascians cells[J]. Biotechnology letters,1989,11(9):653-658.
[20] KITA M,HIRATA Y,MORIGUCHI T,ET A L. Molecular cloning and characterization of a novel gene encoding limonoid UDP glucosyltransferase in Citrus 1[J]. Febs Letters,2000,469(2/3):173-178.
[21] KARIM M R,HASHINAGA F. Preparation and properties of immobilized pummelo limonoid glucosyltransferase[J]. Process Biochemistry,2002,38(5):809-814.
[22]DUA K,KOCHER G S. Enrichment and characterization of limonin degrading microorganisms isolated from kinnow orchard soil,peel and waste site[J]. Applied Biological Research,2017,19(2):187-196.
[23]PURI M,BANERJEE U C. Production,purification and characterization of the debittering enzyme naringinase[J]. Biotechnology Advances,2000,18(3):207-217.
[24]胡阳.蜜柚苦味物的HPLC检测、提取及脱苦菌株的筛选[D].雅安:四川农业大学,2007:36-48.
[25]董晨,张锦华,白宝清,等.柠檬苦素高效降解菌的分离筛选与鉴定[J].食品与发酵工业,2017,43(8):67-72.
[26]纪学芳,师俊玲,张锦华.柠檬苦素降解菌的分离筛选与分类鉴定[J].食品科学,2011,32(15):177-181.
[27]刘青茹,高彦祥.柑桔汁脱苦降酸方法研究进展[J].食品研究与开发,2015,36(13):127-133.
[28]邓丽莉,潘晓倩,生吉平,等.考马斯亮蓝法测定苹果组织微量可溶性蛋白含量的条件优化[J].食品科学,2012,33(24):185-189.
[29]王松林,彭荣,崔榕,等.类柠檬苦素生物转化与脱苦研究进展[J].食品科学,2015,36(9):279-283.
[30] STINCO C M,FERN魣NDEZ-V魣ZQUEZ R,HERNANZ D,et al.Industrial orange juice debittering:Impact on bioactive compounds and nutritional value[J]. Journal of Food Engineering,2013,116(1):155-161.
[31]刘君.柑桔种子中类柠檬苦素提取纯化工艺研究[D].重庆:西南大学,2008:11-13.
[32] HASEGAWA S,PELTON V A,BENNETT R D. Metabolism of limonoids by Arthrobacter globiformis II:basis for a practical means of reducing the limonin content of orange juice by immobilized cells[J]. Journal of Agricultural and Food Chemistry,1983,31(5):1002-1004.
[33] PATIL M B,DHAKE A B. Debittering of citrus fruit juice by naringinase of Penicillium purpurogenum[J]. Int J Engg Res Sci Tech,2014,3:266-270.
[34]高秋芬,倪辉,李利君,等.黑曲霉胞外酶对柑橘果汁的脱苦作用[J].中国食品学报,2014,14(4):156-163.
[35]黄颖颖,陆东和,杨成龙,等.酶法脱苦对琯溪蜜柚汁主要苦味物质及营养风味的影响[J].福建农业学报,2016,31(8):886-891.