出芽短梗霉发酵液中聚苹果酸测定方法的比较研究
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摘要
出芽短梗霉(Aureobasidium pullulans)发酵产聚苹果酸(PMA)的研究近年来受到广泛关注。发酵液中聚苹果酸的测定通常是将聚苹果酸水解成苹果酸后,通过分光光度法(Goodban法)或HPLC法进行测定。本文先对乙醇沉淀法测定聚苹果酸含量进行了研究,发现乙醇沉淀法无法将聚苹果酸选择性地沉淀出来,沉淀物中含有一定量的普鲁兰多糖。因此进一步建立了先乙醇沉淀再分光光度法测定的新方法,即:将乙醇沉淀物重新溶解,经酸水解后再以分光光度法进行测定。试验结果表明,该方法与HPLC法的检测结果比较接近,而且可以免除发酵液中黑色素产生对分光光度法测定的干扰,可以对出芽短梗霉发酵液中的聚苹果酸含量进行准确测定。
The research of fermentative production of poly-malic acid(PMA)by Aureobasidium pullulans has attracted extensive attentions in recent years. The determination of PMA was usually by measuring the amount of malic acid by spectrophotometry or HPLC after hydrolysis. The precipitation of PMA from fermentation broth by ethanol was studied. The results showed that PMA could not be precipitated selectively by ethanol,and certain amount of pullulan was detected in the precipitate. A new method was established by combining precipitation of PMA and spectrophotometry,the process was as follows :after ethanol precipitation,the perecipitate was dissolved in pure water and hydrolysis by H2 SO4,the obtained hydrolysate was used for malic acid detection by spectrophotometry. The study results were very similar to that of HPLC,so the new method could be used for accurate measurement of PMA in Aureobasidium pullulans fermentation broth without concerning of black pigments interfering by spectrophotometer.
The research of fermentative production of poly-malic acid(PMA)by Aureobasidium pullulans has attracted extensive attentions in recent years. The determination of PMA was usually by measuring the amount of malic acid by spectrophotometry or HPLC after hydrolysis. The precipitation of PMA from fermentation broth by ethanol was studied. The results showed that PMA could not be precipitated selectively by ethanol,and certain amount of pullulan was detected in the precipitate. A new method was established by combining precipitation of PMA and spectrophotometry,the process was as follows :after ethanol precipitation,the perecipitate was dissolved in pure water and hydrolysis by H2 SO4,the obtained hydrolysate was used for malic acid detection by spectrophotometry. The study results were very similar to that of HPLC,so the new method could be used for accurate measurement of PMA in Aureobasidium pullulans fermentation broth without concerning of black pigments interfering by spectrophotometer.
引文
[1]司振军.微生物高效生产吡咯喹啉醌和聚苹果酸的研究[D].杭州,浙江大学,2017:99-100.
[2] Ding H, Klibanov A M.Inhibition of brain tumor growth by intravenous poly(β-L-malic acid)nanobioconjugate with pH-dependent drug release[J].Proceedings of the National Academy of Sciences, 2010, 107(42):18143-18148.
[3]戈进杰.生物降解高分子材料及其应用[M].北京:化学工业出版社.2002:41-44.
[4] Guerin P, Vert M, Braund C, et al.Optically active poly(β-malic acid)[J].Polymer Bulletin, 1985, 14(2):187-192.
[5]丁锐,李光吉.生物高分子聚苹果酸及其衍生物的合成与应用前景[J].高分子通报,2005(2):49-58.
[6] Vcrt M.Chemical routes to poly(β-malic acid)and potential applications of this water-soluble bioresorbable poly(β-hydroxy alkanoate)[J]. Polymer Degradation and Stability,1998, 59(1):169-175.
[7] Cammas-Marion S, Bear M M, Harada A, et al. New macromolecular micelles based on degradable amphiphilic block copolymers of malic acid and malic acid ester[J].Macromolecular Chemistry&Physics, 2000, 201(3):355-364.
[8] Holler E.Handbook of Engineering Polymeric Materials:Poly(malic Acid)from Natural Sources[M].Crc Press, New York,1998:93-103.
[9] Kajiyama T, Kobayashi H, Taguchi T, et al.Synthesis of activated poly(α,β-malic acid)using N-hydroxysuccinimide and its gelation with collagen as biomaterials[J]. Materials Science&Engineering C, 2004, 24(6):815-819.
[10] Kajiyama T, Taguchi T, Kobayashi H, et al. Synthesis of high molecular weight poly(α,β-malic acid)for biomedical use by direct polycondensation[J]. Polymer Degradation&Stability, 2003, 81(3):525-530.
[11] Shimada K, Matsushima K, Fukumoto J, et al.Poly-(L)-malic acid; A new protease inhibitor from Penicillium cyclopium[J]. Biochemical&Biophysical Research Communications, 1969, 35(5):619-624.
[12] Fischer H, Erdmann S, Holler E. An unusual polyanion from Physarum polycephalum that inhibits homologous DNA polymerase alpha in vitro[J]. Biochemistry, 1989, 28(12):5219-26.
[13] Nagata N, Nakahara T, Tabuchi T.Fermentative production of poly(β-L-malic acid), a polyelectronic biopolyester, by Aureobasidium sp.[J]. Biosci. Biotech. Biochem., 1993, 57:638-642.
[14] Antonelli M L, Spadaro C, Tornelli R F. A microcalorimetric sensor for food and cosmetic analyses:L-malic acid determination[J]. Talanta, 2008, 74(5):1450-1454.
[15] Posyniak A, Zmudzki J, Mitrowska K.Dispersive solid-phase extraction for the determination of sulfonamides in chicken muscle by liquid chromatography[J]. Journal of Chromatography A, 2005, 1087(1):259-264.
[16]毕艺成,陆宏艳,王浩,等.两步超滤膜法分离提取发酵液中聚苹果酸[J].膜科学与技术,2015, 35(1):97-102.
[17]陈珊,黄艳玲,张乐,等.微生物发酵生产聚苹果酸的测定研究[J].农产品加工月刊,2017(5):49-50.
[18]沈萍,陈向东.微生物学实验[M].北京:高等教育出版社,2007, 14-20.
[19]王凤荣,乔长晟,江建梅,等.出芽短梗霉发酵液中聚苹果酸测定方法的比较[J].食品工业科技,2010, 31(6):347-349.
[20] Braud C, Bunel C, Vert M. Poly(β-malic acid):a new polymeric drug carrier, evidence for degradation in vitro[J].Polymer Bulletin, 1985, 13(4):293-299.
[21] Schmidt A,Windisch C, Holler E.Nuclear accumulation and homeostasis of the unusual polymer beta-poly(L-malate)in plasmodia of Physarum polycephalum[J]. European Journal of Cell Biology, 1996, 70(4):373-380.
[22]郑谊丰,周华,王丽燕,等.高效液相色谱法测定发酵液中聚苹果酸方法的建立[J].食品科技,2010, 35(4):243-246.
[23]张慧莉.微生物高效生产γ-聚谷氨酸和β-聚苹果酸的研究[D].杭州,浙江大学,2012:28-29.
[2] Ding H, Klibanov A M.Inhibition of brain tumor growth by intravenous poly(β-L-malic acid)nanobioconjugate with pH-dependent drug release[J].Proceedings of the National Academy of Sciences, 2010, 107(42):18143-18148.
[3]戈进杰.生物降解高分子材料及其应用[M].北京:化学工业出版社.2002:41-44.
[4] Guerin P, Vert M, Braund C, et al.Optically active poly(β-malic acid)[J].Polymer Bulletin, 1985, 14(2):187-192.
[5]丁锐,李光吉.生物高分子聚苹果酸及其衍生物的合成与应用前景[J].高分子通报,2005(2):49-58.
[6] Vcrt M.Chemical routes to poly(β-malic acid)and potential applications of this water-soluble bioresorbable poly(β-hydroxy alkanoate)[J]. Polymer Degradation and Stability,1998, 59(1):169-175.
[7] Cammas-Marion S, Bear M M, Harada A, et al. New macromolecular micelles based on degradable amphiphilic block copolymers of malic acid and malic acid ester[J].Macromolecular Chemistry&Physics, 2000, 201(3):355-364.
[8] Holler E.Handbook of Engineering Polymeric Materials:Poly(malic Acid)from Natural Sources[M].Crc Press, New York,1998:93-103.
[9] Kajiyama T, Kobayashi H, Taguchi T, et al.Synthesis of activated poly(α,β-malic acid)using N-hydroxysuccinimide and its gelation with collagen as biomaterials[J]. Materials Science&Engineering C, 2004, 24(6):815-819.
[10] Kajiyama T, Taguchi T, Kobayashi H, et al. Synthesis of high molecular weight poly(α,β-malic acid)for biomedical use by direct polycondensation[J]. Polymer Degradation&Stability, 2003, 81(3):525-530.
[11] Shimada K, Matsushima K, Fukumoto J, et al.Poly-(L)-malic acid; A new protease inhibitor from Penicillium cyclopium[J]. Biochemical&Biophysical Research Communications, 1969, 35(5):619-624.
[12] Fischer H, Erdmann S, Holler E. An unusual polyanion from Physarum polycephalum that inhibits homologous DNA polymerase alpha in vitro[J]. Biochemistry, 1989, 28(12):5219-26.
[13] Nagata N, Nakahara T, Tabuchi T.Fermentative production of poly(β-L-malic acid), a polyelectronic biopolyester, by Aureobasidium sp.[J]. Biosci. Biotech. Biochem., 1993, 57:638-642.
[14] Antonelli M L, Spadaro C, Tornelli R F. A microcalorimetric sensor for food and cosmetic analyses:L-malic acid determination[J]. Talanta, 2008, 74(5):1450-1454.
[15] Posyniak A, Zmudzki J, Mitrowska K.Dispersive solid-phase extraction for the determination of sulfonamides in chicken muscle by liquid chromatography[J]. Journal of Chromatography A, 2005, 1087(1):259-264.
[16]毕艺成,陆宏艳,王浩,等.两步超滤膜法分离提取发酵液中聚苹果酸[J].膜科学与技术,2015, 35(1):97-102.
[17]陈珊,黄艳玲,张乐,等.微生物发酵生产聚苹果酸的测定研究[J].农产品加工月刊,2017(5):49-50.
[18]沈萍,陈向东.微生物学实验[M].北京:高等教育出版社,2007, 14-20.
[19]王凤荣,乔长晟,江建梅,等.出芽短梗霉发酵液中聚苹果酸测定方法的比较[J].食品工业科技,2010, 31(6):347-349.
[20] Braud C, Bunel C, Vert M. Poly(β-malic acid):a new polymeric drug carrier, evidence for degradation in vitro[J].Polymer Bulletin, 1985, 13(4):293-299.
[21] Schmidt A,Windisch C, Holler E.Nuclear accumulation and homeostasis of the unusual polymer beta-poly(L-malate)in plasmodia of Physarum polycephalum[J]. European Journal of Cell Biology, 1996, 70(4):373-380.
[22]郑谊丰,周华,王丽燕,等.高效液相色谱法测定发酵液中聚苹果酸方法的建立[J].食品科技,2010, 35(4):243-246.
[23]张慧莉.微生物高效生产γ-聚谷氨酸和β-聚苹果酸的研究[D].杭州,浙江大学,2012:28-29.