采用双水相萃取技术提取ε-聚赖氨酸
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摘要
对以双水相萃取新技术提取ε-聚赖氨酸(ε-poly-L-lysine,ε-PL)作了较深入的探索研究。通过对多个双水相体系的筛选,确定了利用乙醇/硫酸铵双水相体系来萃取ε-PL的方案,得到的最佳萃取条件为:pH 9.5,乙醇/硫酸铵质量分数组成为20%/20%,分配系数和收率分别可以达到18.33和98.02%。进一步探究了多级萃取方式对ε-PL萃取效果的影响,发现3次萃取的效果最好,蛋白质和色素去除率分别达到85.66%和60.00%,ε-PL收率为96.15%。最后,收集经过3次萃取后的上相,利用超滤膜进行简单脱盐浓缩,产品的收率和纯度分别可以达到68.28%和92.39%。
An integrated fermentation-separation process for the production of ε-poly-L-lysine( ε-PL) was investigated. Through the screening of several aqueous two-phase systems,the extraction of ε-PL by ethanol/ammonium sulfate aqueous two-phase system was investigated in detail. The results indicated that the highest partition coefficient(18. 33) and recovery(98. 02%) of ε-PL were obtained with an aqueous two-phase system composed of 20%(w/w) ethanol and 20%(w/w) ammonium sulfate,pH9. 5. The effect of multi-stage extraction was investigated. It was found that the effect of extraction for three times was the best,the removal rate of protein and pigment were 85.66% and 60. 00%,respectively,and the yield of ε-PL was 96. 15%. Finally,the top phase after three times of extraction was desalted and concentrated by ultrafiltration membrane,and the yield and purity of the product reached68. 28% and 92. 39%,respectively.
An integrated fermentation-separation process for the production of ε-poly-L-lysine( ε-PL) was investigated. Through the screening of several aqueous two-phase systems,the extraction of ε-PL by ethanol/ammonium sulfate aqueous two-phase system was investigated in detail. The results indicated that the highest partition coefficient(18. 33) and recovery(98. 02%) of ε-PL were obtained with an aqueous two-phase system composed of 20%(w/w) ethanol and 20%(w/w) ammonium sulfate,pH9. 5. The effect of multi-stage extraction was investigated. It was found that the effect of extraction for three times was the best,the removal rate of protein and pigment were 85.66% and 60. 00%,respectively,and the yield of ε-PL was 96. 15%. Finally,the top phase after three times of extraction was desalted and concentrated by ultrafiltration membrane,and the yield and purity of the product reached68. 28% and 92. 39%,respectively.
引文
[1]SHIMA S,SAKAI H.Poly-L-lysine produced by Streptomyces.Part III.chemical studies[J].Journal of the Agricultural and Biological Chemistry Society of Japan,1981,45(11):2 497-2 502.
[2]MORTEN H,TINA M,VAD B S,et al.The antimicrobial mechanism of action of epsilon-poly-l-lysine[J].Applied and Environmental Microbiology,2014,80(24):7 758-7 770.
[3]US Food and Drug Administration.GRN 000135-2004bε-poly-lysine for addition to specified foods[S].Washington D.C.U.S.Office of Food additive Safty,Accessed 27 Jul2005.
[4]甄斌.ε-聚赖氨酸高效提取工艺构建:固液分离与脱色工艺研究[D].无锡:江南大学,2015.
[5]周加祥,刘铮.生物分离技术与过程研究进展[J].化工进展,2000,19(6):38-41.
[6]马春宏,朱红,王良,等.双水相萃取技术的应用研究进展[J].光谱实验室,2010,27(5):1 906-1 914.
[7]CHEN X S,LI S,LIAO L J,et al.Production ofε-polyL:-lysine using a novel two-stage p H control strategy by Streptomyces sp.M-Z18 from glycerol[J].Bioprocess Biosyst Eng,2011,34(5):561-567.
[8]LI Z G,SUN Y Q,ZHENG W L,et al.A novel and environment-friendly bioprocess of 1,3-propanediol fermentation integrated with aqueous two-phase extraction by ethanol/sodium carbonate system[J].Biochemical Engineering Journal,2013,80(7):68-75.
[9]WU Y,WANG Y,ZHANG W,et al.Extraction and preliminary purification of anthocyanins from grape juice in aqueous two-phase system[J].Separation&Purification Technology,2014,124(6):170-178.
[10]谢志鹏,徐志南,郑建明,等.靛酚蓝反应测定发酵液中的氨态氮[J].浙江大学学报(工学版),2005,39(3):437-439+444.
[11]王柯.酒精—沼气双发酵耦联生态系统的机理和关键技术研究[D].无锡:江南大学,2014.
[12]CHANDRASEKHAR J,SONIKA G,MADHUSUDHAN M C,et al.Differential partitioning of betacyanins and betaxanthins employing aqueous two phase extraction[J].Journal of Food Engineering,2015,144:156-163.
[13]ITZHAKI R F.Colorimetric method for estimating polylysine and polyarginine[J].Analytical Biochemistry,1972,50(2):569-574.
[14]范宝庆,贾士儒,戴玉杰,等.微生物发酵法产ε-聚赖氨酸的质量分析[J].中国食品添加剂,2010(3):108-112.
[15]BRADFORD M M.A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[J].Analytical Biochemistry,1976,72(s 1-2):248-254.
[16]ZHEN Bin,CHEN Xu-sheng,HAN Dai,et al.An alternative method for the decoloration ofε-poly-l-lysine eluate by macroporous resin in the separation and purification ofε-poly-l-lysine from fermentation broth[J].Food&Bioproducts Processing,2015,95:332-338.
[17]LI Z,JIANG B,ZHANG D,et al.Aqueous two-phase extraction of 1,3-propanediol from glycerol-based fermentation broths[J].Separation&Purification Technology,2009,66(3):472-478.
[18]李丽敏,尚庆坤.亲水有机溶剂/无机盐双水相萃取技术在分离提纯生物物质中的应用[J].光谱实验室,2011,28(6):3 073-3 078.
[19]HIRAKI J,ICHIKAWA T,NINOMIVA S I,et al.Use of ADME studies to confirm the safety ofε-polylysine as a preservative in food[J].Regulatory Toxicology&Pharmacology Rtp,2003,37(2):328-340.
[20]毛忠贵.生物工业下游技术[M].北京:中国轻工业出版社,1900:80-98.
[21]陈旭升,艾婷婷,甑斌,等.ε-聚赖氨酸提取工艺中活性炭脱色条件研究[J].中国食品添加剂,2013(5):99-103.
[22]艾婷婷,陈旭升,李树,等.冷冻及加热法去除ε-聚赖氨酸发酵液中的蛋白[J].食品工业科技,2012,33(19):236-238;242.
[23]刘杨,虞永蕾,苏红彩,等.成相无机盐种类对PEG双水相相平衡影响[J].化学研究与应用,2011,23(9):1 205-1 208.
[2]MORTEN H,TINA M,VAD B S,et al.The antimicrobial mechanism of action of epsilon-poly-l-lysine[J].Applied and Environmental Microbiology,2014,80(24):7 758-7 770.
[3]US Food and Drug Administration.GRN 000135-2004bε-poly-lysine for addition to specified foods[S].Washington D.C.U.S.Office of Food additive Safty,Accessed 27 Jul2005.
[4]甄斌.ε-聚赖氨酸高效提取工艺构建:固液分离与脱色工艺研究[D].无锡:江南大学,2015.
[5]周加祥,刘铮.生物分离技术与过程研究进展[J].化工进展,2000,19(6):38-41.
[6]马春宏,朱红,王良,等.双水相萃取技术的应用研究进展[J].光谱实验室,2010,27(5):1 906-1 914.
[7]CHEN X S,LI S,LIAO L J,et al.Production ofε-polyL:-lysine using a novel two-stage p H control strategy by Streptomyces sp.M-Z18 from glycerol[J].Bioprocess Biosyst Eng,2011,34(5):561-567.
[8]LI Z G,SUN Y Q,ZHENG W L,et al.A novel and environment-friendly bioprocess of 1,3-propanediol fermentation integrated with aqueous two-phase extraction by ethanol/sodium carbonate system[J].Biochemical Engineering Journal,2013,80(7):68-75.
[9]WU Y,WANG Y,ZHANG W,et al.Extraction and preliminary purification of anthocyanins from grape juice in aqueous two-phase system[J].Separation&Purification Technology,2014,124(6):170-178.
[10]谢志鹏,徐志南,郑建明,等.靛酚蓝反应测定发酵液中的氨态氮[J].浙江大学学报(工学版),2005,39(3):437-439+444.
[11]王柯.酒精—沼气双发酵耦联生态系统的机理和关键技术研究[D].无锡:江南大学,2014.
[12]CHANDRASEKHAR J,SONIKA G,MADHUSUDHAN M C,et al.Differential partitioning of betacyanins and betaxanthins employing aqueous two phase extraction[J].Journal of Food Engineering,2015,144:156-163.
[13]ITZHAKI R F.Colorimetric method for estimating polylysine and polyarginine[J].Analytical Biochemistry,1972,50(2):569-574.
[14]范宝庆,贾士儒,戴玉杰,等.微生物发酵法产ε-聚赖氨酸的质量分析[J].中国食品添加剂,2010(3):108-112.
[15]BRADFORD M M.A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[J].Analytical Biochemistry,1976,72(s 1-2):248-254.
[16]ZHEN Bin,CHEN Xu-sheng,HAN Dai,et al.An alternative method for the decoloration ofε-poly-l-lysine eluate by macroporous resin in the separation and purification ofε-poly-l-lysine from fermentation broth[J].Food&Bioproducts Processing,2015,95:332-338.
[17]LI Z,JIANG B,ZHANG D,et al.Aqueous two-phase extraction of 1,3-propanediol from glycerol-based fermentation broths[J].Separation&Purification Technology,2009,66(3):472-478.
[18]李丽敏,尚庆坤.亲水有机溶剂/无机盐双水相萃取技术在分离提纯生物物质中的应用[J].光谱实验室,2011,28(6):3 073-3 078.
[19]HIRAKI J,ICHIKAWA T,NINOMIVA S I,et al.Use of ADME studies to confirm the safety ofε-polylysine as a preservative in food[J].Regulatory Toxicology&Pharmacology Rtp,2003,37(2):328-340.
[20]毛忠贵.生物工业下游技术[M].北京:中国轻工业出版社,1900:80-98.
[21]陈旭升,艾婷婷,甑斌,等.ε-聚赖氨酸提取工艺中活性炭脱色条件研究[J].中国食品添加剂,2013(5):99-103.
[22]艾婷婷,陈旭升,李树,等.冷冻及加热法去除ε-聚赖氨酸发酵液中的蛋白[J].食品工业科技,2012,33(19):236-238;242.
[23]刘杨,虞永蕾,苏红彩,等.成相无机盐种类对PEG双水相相平衡影响[J].化学研究与应用,2011,23(9):1 205-1 208.