外源添加氨基酸对兽疫链球菌产透明质酸的影响
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摘要
兽疫链球菌(Streptococcus zooepidemicus)是一种营养缺陷型菌株,其生长代谢需要外源添加各类营养因子。通过在兽疫链球菌发酵对数生长初期外源添加氨基酸的方式研究其对透明质酸生产的影响。结果表明,外源添加氨基酸对透明质酸的生产具有显著的促进作用。笔者初步研究了当添加质量浓度为0.5 g/L时,18种常见氨基酸对透明质酸产量的影响。在此基础上,在18种氨基酸中选取苯丙氨酸和丝氨酸对透明质酸合成进行浓度优化实验,结合细胞生长和代谢产物分析,更进一步研究氨基酸对透明质酸合成的影响。结果表明,苯丙氨酸和丝氨酸添加质量浓度分别为0.5 g/L和0.5 g/L时,透明质酸产量提高最大,与对照相比分别提高了18.05%和17.44%,菌体浓度均没有增加,但是乳酸浓度分别降低了9.93%和15.80%,说明氨基酸的添加不是通过增加菌体量来提高透明质酸产量,而是改变了碳源流向,减少了乳酸积累,使得透明质酸产量显著提高。本研究为高产透明质酸的发酵工艺提供了借鉴与依据。
Streptococcus zooepidemicus is an auxotrophic strain,which requires the exogenous addition of various nutrient factorsduring their growth and metabolism. In this study,the effect of exogenous amino acid addition in the early stage of the logarithmic growth of Streptococcus zooepidemicus on the production of hyaluronic acid was investigated.The results showed that the exogenously added amino acids had a significant effect on the production of hyaluronic acid. The authors initially explored the effect of 18 common amino acids on the production of hyaluronic acid when the mass concentration was 0.5 g/L.Studies found that the exogenous addition of phenylalanine and serine had a strong promoting effect on HA synthesis,while the exogenous addition of threonine and tyrosine had a certain inhibitory effect on HA synthesis. On this basis,phenylalanine and serine were selected from 18 amino acids for concentration optimization experiments of hyaluronic acid synthesis. Lactic acid was the main by-product of hyaluronic acid synthesis.Hyaluronic acid was the main component of bacterial capsules. Therefore,combined with cell growth and lactic acid metabolism analysis,the effect of amino acids on hyaluronic acid synthesis was further investigated. The results showed that,when the mass concentrations of phenylalanine and serine were 0.5 g/L and 0.5 g/L,respectively,the production of hyaluronic acid was increased by 18.05% and 17.44%,respectively,compared with the control. At this case,the concentration of the bacteria did not increase,but the concentration of lactic acid decreased by 9.93% and15.80%,respectively,indicating that the addition of amino acids did not increase the yield of hyaluronic acid by increasing the bacteria,but changed the flow of carbon sources and reduced the accumulation of lactic acid. The carbon source flowed to the direction of hyaluronic acid synthesis,thereby increasing its yield. At the same time,it was found that the addition of amino acid did not change the consumption rate of glucose but increased the rate of hyaluronic acid synthesis. After the addition of phenylalanine,the optimal fermentation time of hyaluronic acid decreased from the original 10 h to 8 h,greatly shortening the fermentation cycle. In this study,amino acids were added exogenously during the fermentation period of auxotrophic strains to investigate its effect on the fermentation of hyaluronic acid,providing reference for the fermentation process of high-yield hyaluronic acid.
Streptococcus zooepidemicus is an auxotrophic strain,which requires the exogenous addition of various nutrient factorsduring their growth and metabolism. In this study,the effect of exogenous amino acid addition in the early stage of the logarithmic growth of Streptococcus zooepidemicus on the production of hyaluronic acid was investigated.The results showed that the exogenously added amino acids had a significant effect on the production of hyaluronic acid. The authors initially explored the effect of 18 common amino acids on the production of hyaluronic acid when the mass concentration was 0.5 g/L.Studies found that the exogenous addition of phenylalanine and serine had a strong promoting effect on HA synthesis,while the exogenous addition of threonine and tyrosine had a certain inhibitory effect on HA synthesis. On this basis,phenylalanine and serine were selected from 18 amino acids for concentration optimization experiments of hyaluronic acid synthesis. Lactic acid was the main by-product of hyaluronic acid synthesis.Hyaluronic acid was the main component of bacterial capsules. Therefore,combined with cell growth and lactic acid metabolism analysis,the effect of amino acids on hyaluronic acid synthesis was further investigated. The results showed that,when the mass concentrations of phenylalanine and serine were 0.5 g/L and 0.5 g/L,respectively,the production of hyaluronic acid was increased by 18.05% and 17.44%,respectively,compared with the control. At this case,the concentration of the bacteria did not increase,but the concentration of lactic acid decreased by 9.93% and15.80%,respectively,indicating that the addition of amino acids did not increase the yield of hyaluronic acid by increasing the bacteria,but changed the flow of carbon sources and reduced the accumulation of lactic acid. The carbon source flowed to the direction of hyaluronic acid synthesis,thereby increasing its yield. At the same time,it was found that the addition of amino acid did not change the consumption rate of glucose but increased the rate of hyaluronic acid synthesis. After the addition of phenylalanine,the optimal fermentation time of hyaluronic acid decreased from the original 10 h to 8 h,greatly shortening the fermentation cycle. In this study,amino acids were added exogenously during the fermentation period of auxotrophic strains to investigate its effect on the fermentation of hyaluronic acid,providing reference for the fermentation process of high-yield hyaluronic acid.
引文
[1]张琳培.微生物发酵优化透明质酸生产的研究进展[J].轻工科技,2018,34(3):18-20,30.ZHANG L P.Research progress in optimization of hyaluronic acid production by microbial fermentation[J].Light Industry Science and Technology,2018,34(3):18-20,30.
[2]BORZACCHIELLO A,GLORIA A,MAYOL L,et al.Natural/synthetic porous scaffold designs and properties for fibro-cartilaginous tissue engineering[J].Journal of Bioactive and Compatible Polymers,2011,26(5):437-451.DOI:10.1177/0883911511420149.
[3]RADAEVA I F,KOSTINA G A,ZMIEVSKII A V.Hyaluronic acid:biological role,structure,synthesis,isolation,purification,and application(review)[J].Prikladnaia Biokhimiia i Mikrobiologiia,1997,33(2):133-137.
[4]LAPCˇIK L,LAPCˇIK L,DE SMEDT S,et al.Hyaluronan:preparation,structure,properties,and applications[J].Chemical Reviews,1998,98(8):2663-2684.DOI:10.1021/cr941199z.
[5]CHONG B F,BLANK L M,MCLAUGHLIN R,et al.Microbial hyaluronic acid production[J].Applied Microbiology and Biotechnology,2005,66(4):341-351.DOI:10.1007/s00253-004-1774-4.
[6]SHIEDLIN A,BIGELOW R,CHRISTOPHER W,et al.Evaluation of hyaluronan from different sources:Streptococcus zooepidemicus,rooster comb,bovine vitreous,and human umbilical cord[J].Biomacromolecules,2004,5(6):2122-2127.DOI:10.1021/bm0498427.
[7]郭学平,王春喜,崔大鹏.发酵法制备透明质酸[J].日用化学工业,1994,24(2):47-48.DOI:10.13218/j.cnki.csdc.1994.02.012.GUO X P,WANG C X,CUI D P.Preparation of hyaluronic acid by fermentation[J].China Surfactant Detergent&Cosmetics,1994,24(2):47-48.
[8]邓静,石岩昌,吴华昌,等.氧载体正己烷对兽疫链球菌发酵生产透明质酸的影响[J].中国医药工业杂志,2009,40(8):578-580.DOI:10.3969/j.issn.1001-8255.2009.08.005.DENG J,SHI Y C,WU H C,et al.Effect of oxygen-carrier nhexane on the production of hyaluronic acid by Streptococcus zooepidemicus[J].Chinese Journal of Pharmaceuticals,2009,40(8):578-580.
[9]管世敏,荣绍丰,陈奕涵,等.外源氧化胁迫提高透明质酸分子量[J].中国医药工业杂志,2013,44(1):27-31.GUAN S M,RONG S F,CHEN Y H,et al.Oxidative stress to increase hyaluranic acid molecule[J].Chinese Journal of Pharmaceuticals,2013,44(1):27-31.
[10]温琦,刘登如,陈坚,等.添加表面活性剂促进兽疫链球菌高产透明质酸[J].化工进展,2006,25(9):1089-1094.DOI:10.3321/j.issn:1000-6613.2006.09.021.WEN Q,LIU D R,CHEN J,et al.Enhancement of hyaluronic acid production by Streptococcus zooepidemicus H24 through supplement of CTAB[J].Chemical Industry and Engineering Progress,2006,25(9):1089-1094.
[11]石岩昌,邓静,吴华昌,等.添加有机前体物质对发酵生产HA的影响研究[J].山东食品发酵,2009(1):7-12.SHI Y C,DENG J,WU H C,et al.The effecting study of producing HA in fermentation by adding differentence organic precondition substance[J].Shandong Food Ferment,2009(1):7-12.
[12]WILLETT N P,MORSE G E,CARLISLE S A.Requirements for growth of Streptococcus agalactiae in a chemically defined medium[J].Journal of Bacteriology,1967,94(4):1247-1248.
[13]刘龙.兽疫链球菌发酵生产透明质酸过程控制与优化[D].无锡:江南大学,2009.LIU L.Process control and optimization of hyaluronic acid production by Streptococcus zooepidemicus[D].Wuxi:Jiangnan University,2009.
[14]高海军,陈坚,章燕芳,等.营养条件对兽疫链球菌发酵生产透明质酸的影响[J].生物工程学报,2000,16(3):396-399.DOI:10.3321/j.issn:1000-3061.2000.03.023.GAO H J,CHEN J,ZHANG Y F,et al.Nutrition condition of hyaluronic acid fermentation with Streptococcus zooepidemicus[J].Chinese Journal of Biotechnology,2000,16(3):396-399.
[15]BITTER T,MUIR H M.A modified uronic acid carbazole reaction[J].Analytical Biochemistry,1962,4(4):330-334.DOI:10.1016/0003-2697(62)90095-7.
[16]CHONG B F,NIELSEN L K.Aerobic cultivation of Streptococcus zooepidemicus and the role of NADH oxidase[J].Biochemical Engineering Journal,2003,16(2):153-162.DOI:10.1016/s1369-703x(03)00031-7.
[17]CHONG B F,BLANK L M,MCLAUGHLIN R,et al.Microbial hyaluronic acid production[J].Applied Microbiology and Biotechnology,2005,66(4):341-351.DOI:10.1007/s00253-004-1774-4.
[18]LIU L,WANG M,DU G,et al.Enhanced hyaluronic acid production of Streptococcus zooepidemicus by an intermittent alkalinestress strategy[J].Letters in Applied Microbiology,2008,46(3):383-388.DOI:10.1111/j.1472-765x.2008.02325.x.
[19]ARMSTRONG D C,COONEY M J,JOHNS M R.Growth and amino acid requirements of hyaluronic-acid-producing Streptococcus zooepidemicus[J].Applied Microbiology and Biotechnology,1997,47(3):309-312.DOI:10.1007/s002530050932.
[2]BORZACCHIELLO A,GLORIA A,MAYOL L,et al.Natural/synthetic porous scaffold designs and properties for fibro-cartilaginous tissue engineering[J].Journal of Bioactive and Compatible Polymers,2011,26(5):437-451.DOI:10.1177/0883911511420149.
[3]RADAEVA I F,KOSTINA G A,ZMIEVSKII A V.Hyaluronic acid:biological role,structure,synthesis,isolation,purification,and application(review)[J].Prikladnaia Biokhimiia i Mikrobiologiia,1997,33(2):133-137.
[4]LAPCˇIK L,LAPCˇIK L,DE SMEDT S,et al.Hyaluronan:preparation,structure,properties,and applications[J].Chemical Reviews,1998,98(8):2663-2684.DOI:10.1021/cr941199z.
[5]CHONG B F,BLANK L M,MCLAUGHLIN R,et al.Microbial hyaluronic acid production[J].Applied Microbiology and Biotechnology,2005,66(4):341-351.DOI:10.1007/s00253-004-1774-4.
[6]SHIEDLIN A,BIGELOW R,CHRISTOPHER W,et al.Evaluation of hyaluronan from different sources:Streptococcus zooepidemicus,rooster comb,bovine vitreous,and human umbilical cord[J].Biomacromolecules,2004,5(6):2122-2127.DOI:10.1021/bm0498427.
[7]郭学平,王春喜,崔大鹏.发酵法制备透明质酸[J].日用化学工业,1994,24(2):47-48.DOI:10.13218/j.cnki.csdc.1994.02.012.GUO X P,WANG C X,CUI D P.Preparation of hyaluronic acid by fermentation[J].China Surfactant Detergent&Cosmetics,1994,24(2):47-48.
[8]邓静,石岩昌,吴华昌,等.氧载体正己烷对兽疫链球菌发酵生产透明质酸的影响[J].中国医药工业杂志,2009,40(8):578-580.DOI:10.3969/j.issn.1001-8255.2009.08.005.DENG J,SHI Y C,WU H C,et al.Effect of oxygen-carrier nhexane on the production of hyaluronic acid by Streptococcus zooepidemicus[J].Chinese Journal of Pharmaceuticals,2009,40(8):578-580.
[9]管世敏,荣绍丰,陈奕涵,等.外源氧化胁迫提高透明质酸分子量[J].中国医药工业杂志,2013,44(1):27-31.GUAN S M,RONG S F,CHEN Y H,et al.Oxidative stress to increase hyaluranic acid molecule[J].Chinese Journal of Pharmaceuticals,2013,44(1):27-31.
[10]温琦,刘登如,陈坚,等.添加表面活性剂促进兽疫链球菌高产透明质酸[J].化工进展,2006,25(9):1089-1094.DOI:10.3321/j.issn:1000-6613.2006.09.021.WEN Q,LIU D R,CHEN J,et al.Enhancement of hyaluronic acid production by Streptococcus zooepidemicus H24 through supplement of CTAB[J].Chemical Industry and Engineering Progress,2006,25(9):1089-1094.
[11]石岩昌,邓静,吴华昌,等.添加有机前体物质对发酵生产HA的影响研究[J].山东食品发酵,2009(1):7-12.SHI Y C,DENG J,WU H C,et al.The effecting study of producing HA in fermentation by adding differentence organic precondition substance[J].Shandong Food Ferment,2009(1):7-12.
[12]WILLETT N P,MORSE G E,CARLISLE S A.Requirements for growth of Streptococcus agalactiae in a chemically defined medium[J].Journal of Bacteriology,1967,94(4):1247-1248.
[13]刘龙.兽疫链球菌发酵生产透明质酸过程控制与优化[D].无锡:江南大学,2009.LIU L.Process control and optimization of hyaluronic acid production by Streptococcus zooepidemicus[D].Wuxi:Jiangnan University,2009.
[14]高海军,陈坚,章燕芳,等.营养条件对兽疫链球菌发酵生产透明质酸的影响[J].生物工程学报,2000,16(3):396-399.DOI:10.3321/j.issn:1000-3061.2000.03.023.GAO H J,CHEN J,ZHANG Y F,et al.Nutrition condition of hyaluronic acid fermentation with Streptococcus zooepidemicus[J].Chinese Journal of Biotechnology,2000,16(3):396-399.
[15]BITTER T,MUIR H M.A modified uronic acid carbazole reaction[J].Analytical Biochemistry,1962,4(4):330-334.DOI:10.1016/0003-2697(62)90095-7.
[16]CHONG B F,NIELSEN L K.Aerobic cultivation of Streptococcus zooepidemicus and the role of NADH oxidase[J].Biochemical Engineering Journal,2003,16(2):153-162.DOI:10.1016/s1369-703x(03)00031-7.
[17]CHONG B F,BLANK L M,MCLAUGHLIN R,et al.Microbial hyaluronic acid production[J].Applied Microbiology and Biotechnology,2005,66(4):341-351.DOI:10.1007/s00253-004-1774-4.
[18]LIU L,WANG M,DU G,et al.Enhanced hyaluronic acid production of Streptococcus zooepidemicus by an intermittent alkalinestress strategy[J].Letters in Applied Microbiology,2008,46(3):383-388.DOI:10.1111/j.1472-765x.2008.02325.x.
[19]ARMSTRONG D C,COONEY M J,JOHNS M R.Growth and amino acid requirements of hyaluronic-acid-producing Streptococcus zooepidemicus[J].Applied Microbiology and Biotechnology,1997,47(3):309-312.DOI:10.1007/s002530050932.