乳链菌肽产生菌Lactococcus lacits subsp. Lactis的发酵工艺研究
|
摘要
乳链菌肽(Nisin)亦称为乳酸链球菌素,是由乳酸乳球菌产生的一种小分子多肽抗菌物质,它可以抑制几乎所有革兰氏阳性菌的生长。由于它对人体无毒,并在酸性条件下极其稳定,因而被公认为天然食品防腐剂。
本文探讨了工业废料在乳酸乳球菌发酵中的应用。通过单因素实验对影响乳链菌肽收率的培养基组分玉米浆、糖蜜、K2HPO4和MgSO4的浓度进行了筛选,实验表明,当糖蜜为30.0 g/L、玉米浆为200.0 g/L、K2HPO4为9.0 g/L、MgSO4为1.0 g/L时,乳链菌肽收率较高。应用Box-Behnken设计得到乳链菌肽最佳发酵培养基配方为:玉米浆243.35 g/L,糖蜜26.77 g/L,MgSO40.44 g/L,K2HPO48.56 g/L时。在此培养条件下,乳链菌肽的收率为1316.0IU/mL。
本文对乳链菌肽普通发酵培养基及培养条件优化进行了优化。利用单因素实验筛选了乳酸乳球菌发酵培养基的成分。在单因素实验的基础上利用Plackett–Burman实验对影响乳链球菌发酵产生乳酸链菌肽的培养基组分进行关键因素筛选,发现酵母浸粉、葡萄糖和K2HPO4为显著影响因素。利用响应面建立了影响乳酸菌肽产率的二次多项数学模型,探讨因素间的交互作用。由实验结果得最优培养基成分为:葡萄糖24.80 g/L,酵母浸粉22.35 g/L, K2HPO413.65 g/L,采用优化培养基得到最优乳链菌肽得率为2115.0 IU/mL。
通过单因素对乳链菌肽发酵过程中的pH值、接种量、转速和温度进行优化,实验结果表明最佳的培养条件为:培养温度36.0℃,摇床转速160r/min,接种量为5%,培养基pH值为7.0。
对乳酸乳球菌进行了发酵罐扩大培养,分别进行了好氧分批发酵和厌氧分批发酵的研究。从实验结果知,氧气有助于乳酸乳球菌的生长,在溶氧维持在30 %时,乳酸乳球菌细胞收率是厌氧条件下的4.92倍。在有氧条件下,乳链菌肽收率略高于无氧发酵的收率。
Nisin, a polypeptide produced by Lactococcus lactis, restrains most Gram-positive bacteria , especially spores of bacterium. Nisin is cosidered as a safe preservative because of its non-toxic and stability at low pH. The study on Nisin began at 1950s and there are more than 50 countries which permit Nisin as preservative. The standard of Nisin was issued in China these years.
The industrial wastes were used as the medium components in the fermentation of Lactococcus lacits subsp.Lactis CGMCC1.2030. It is shown that Nisin productivity was mainly influenced by corn steep liquor, molasses,K2HPO4 and MgSO4 with single factor experiment and the optimal levels were 200 g/L, 30 g/L, 9 g/L and 1 g/L respectively. Box-Behnken design and response surface analysis methods were applied to confirm optimum concentration of mainly effect factor.The results showed that the optimum medium culture was corn steep liquor 243.35 g/L,molasses 26.77 g/L,K2HPO4 8.56 g/L,MgSO4 0.44 g/L.The predictive Nisin productivity is 1334 IU/mL with optimal medium. The validation experiments with optimal medium were conducted 5 times and the average Nisin productivity is 1315.6IU/mL, which confirm the model could fit the fermentation conditions. The cost could be decreased significantly by the industrial waste as medium components to produce Nisin.
With the Plackett-Burman method , the factors affected the Nisin titer were studied , and three important components were got , which are glucose , yeast extract and K2HPO4. Box-Behnken experiment were used to optimize the medium , the optimal medium were got as : glucose 24.80 g/L , yeast extract 22.35 g/L,K2HPO4 13.65 g/L. Under the optimal condition , the max predicted Nisin titer is 2157 IU/mL ,while the experimental results 2115 IU/mL .
The effects of the medium pH,temperature,rotate speed,inoculum size were studied in this work . The result suggests the optimal culture condition as : medium pH 7 , temperature 36℃, rotate speed 160 r/min , inoculum size 5 %.
The pilot-scale fermentation was carried in the Biomate 10 L fermenter . Anaerobic and aerobic batch fermentation was studied. The biomass and Nisin titer were higher in aerobie fermentation. The biomass with aerobic batch fermentation were 4.92 times compared with anaerobic fermentation,while the Nisin titer of aerobic batch fermentation was little higher than that of anaerobic fermentation.
本文探讨了工业废料在乳酸乳球菌发酵中的应用。通过单因素实验对影响乳链菌肽收率的培养基组分玉米浆、糖蜜、K2HPO4和MgSO4的浓度进行了筛选,实验表明,当糖蜜为30.0 g/L、玉米浆为200.0 g/L、K2HPO4为9.0 g/L、MgSO4为1.0 g/L时,乳链菌肽收率较高。应用Box-Behnken设计得到乳链菌肽最佳发酵培养基配方为:玉米浆243.35 g/L,糖蜜26.77 g/L,MgSO40.44 g/L,K2HPO48.56 g/L时。在此培养条件下,乳链菌肽的收率为1316.0IU/mL。
本文对乳链菌肽普通发酵培养基及培养条件优化进行了优化。利用单因素实验筛选了乳酸乳球菌发酵培养基的成分。在单因素实验的基础上利用Plackett–Burman实验对影响乳链球菌发酵产生乳酸链菌肽的培养基组分进行关键因素筛选,发现酵母浸粉、葡萄糖和K2HPO4为显著影响因素。利用响应面建立了影响乳酸菌肽产率的二次多项数学模型,探讨因素间的交互作用。由实验结果得最优培养基成分为:葡萄糖24.80 g/L,酵母浸粉22.35 g/L, K2HPO413.65 g/L,采用优化培养基得到最优乳链菌肽得率为2115.0 IU/mL。
通过单因素对乳链菌肽发酵过程中的pH值、接种量、转速和温度进行优化,实验结果表明最佳的培养条件为:培养温度36.0℃,摇床转速160r/min,接种量为5%,培养基pH值为7.0。
对乳酸乳球菌进行了发酵罐扩大培养,分别进行了好氧分批发酵和厌氧分批发酵的研究。从实验结果知,氧气有助于乳酸乳球菌的生长,在溶氧维持在30 %时,乳酸乳球菌细胞收率是厌氧条件下的4.92倍。在有氧条件下,乳链菌肽收率略高于无氧发酵的收率。
Nisin, a polypeptide produced by Lactococcus lactis, restrains most Gram-positive bacteria , especially spores of bacterium. Nisin is cosidered as a safe preservative because of its non-toxic and stability at low pH. The study on Nisin began at 1950s and there are more than 50 countries which permit Nisin as preservative. The standard of Nisin was issued in China these years.
The industrial wastes were used as the medium components in the fermentation of Lactococcus lacits subsp.Lactis CGMCC1.2030. It is shown that Nisin productivity was mainly influenced by corn steep liquor, molasses,K2HPO4 and MgSO4 with single factor experiment and the optimal levels were 200 g/L, 30 g/L, 9 g/L and 1 g/L respectively. Box-Behnken design and response surface analysis methods were applied to confirm optimum concentration of mainly effect factor.The results showed that the optimum medium culture was corn steep liquor 243.35 g/L,molasses 26.77 g/L,K2HPO4 8.56 g/L,MgSO4 0.44 g/L.The predictive Nisin productivity is 1334 IU/mL with optimal medium. The validation experiments with optimal medium were conducted 5 times and the average Nisin productivity is 1315.6IU/mL, which confirm the model could fit the fermentation conditions. The cost could be decreased significantly by the industrial waste as medium components to produce Nisin.
With the Plackett-Burman method , the factors affected the Nisin titer were studied , and three important components were got , which are glucose , yeast extract and K2HPO4. Box-Behnken experiment were used to optimize the medium , the optimal medium were got as : glucose 24.80 g/L , yeast extract 22.35 g/L,K2HPO4 13.65 g/L. Under the optimal condition , the max predicted Nisin titer is 2157 IU/mL ,while the experimental results 2115 IU/mL .
The effects of the medium pH,temperature,rotate speed,inoculum size were studied in this work . The result suggests the optimal culture condition as : medium pH 7 , temperature 36℃, rotate speed 160 r/min , inoculum size 5 %.
The pilot-scale fermentation was carried in the Biomate 10 L fermenter . Anaerobic and aerobic batch fermentation was studied. The biomass and Nisin titer were higher in aerobie fermentation. The biomass with aerobic batch fermentation were 4.92 times compared with anaerobic fermentation,while the Nisin titer of aerobic batch fermentation was little higher than that of anaerobic fermentation.
引文
[1].K,A.Setvens,B.W.Sheldon,N.A.KlaPes,eta1.NisintreatmentforinaetivationofSalmone11a sPeeiesandothergram一negativebaeteria.ApplEnvironMierobiol,1991,57:3613-3615
[2] Rogers LA, whiter EO. Limiting Factors in the lactic fermentation [J]. Journal of Bacteriology. 1928,16:211- 229.
[3] Mattick A T R, Hirsch A. A Powerful inhibitory Substance produced by group N Streptococci [J]. Nature(London).1944,154:551.
[4] de Vos WM, Mulders JW, Siezen RJ, et al. Properties ofNisinZ and distribution of its gene,nisZ, in Lactococcus lactis [J ] . Applied and Environmental Microbiology. 59 (1): 213 -218.
[5] Morihiko Meeda, Takashi Iwashita Yoshie Kurihara. Studies on Modifiers. .ⅡPurification and Structure Determination of Grmnemic Acids, Antisweet Active Principle from Grmnema. Sylvestre Leaves[J].Tetrahedron Letters.1989, 30(12):1547-1550.
[6]还连栋,陈秀珠,才迎,等.乳链菌肽产生菌的定向筛选及发酵产物的鉴定[J].微生物学报.1997, 37(4):292-300.
[7]庄绪亮,马桂荣.乳链菌肽的特殊结构与功能[J] .生命的化学.1998, 18 (2):37– 39.
[8]. LLin,PDuearme,EBreukink,etal.ComPutationalstudyofNisininteraetionwithmodel membrane.BioehimBiophysaeta,1999,420(1):111一120
[9].EADavies,HEBevis,JDBroughton.TheuseofthebaeterioeinNisin,asapreservatives inrieott--atyPeeheesestoeontrolthefood一bomePathogenListeriamonoeytogenes.APPI Mierobiol,1997,24:343一346
[10]陈刚新,谢红国,杨丰科.发酵工艺代谢调控[J].化学与生物工程2005, 5:9-11
[11]孔保华,Nisin在红肠保鲜中的应用,酿酒科技,1999(2):38-44.
[12]袁秋萍,乳酸链球菌素在肉制品中的应用, 1998(04):27-28.
[13]汤凤霞,蔡慧农.微生物防腐剂Nisin的研究进展[J].世界农业.2003,(2):43-44.
[14]郭清泉,张兰威,冯镇等.Nisin在酸奶制品中的应用[J].食品与机械.2001,(4):30-31.
[15] Radler. Local measurements of viscoelastic moduli of entangled actin networks using an oscillating magnetic bead micro-rheometer. Biophys J. 1994,66(6): 2210–2216.
[16]庄绪亮,张洪勋,马桂荣,等.乳链菌肽发酵的主要影响因子.化工冶金,2000,21(1):93-97
[17]吴琼,吕红线,王锦,等.乳链菌肽高产菌株发酵条件的研究.工业微生物,2000,30(1):36-39
[18]侯革非,何仁.乳酸链球菌素抑菌效果的研究广西工学院学报,2001,12(4):63-65
[19]庄绪亮.乳链菌肽发酵的主要影响因子[J].化工冶金,2000,21(1):93-97
[20]李孱,白景华.细菌素发酵培养基的优化及动力学初步分析[J].生物工程学报,2001,17(2):187-192
[21]季伟,徐学明.乳酸链球菌固态发酵豆粨的研究[J].饲料资源开发与利用,2006,5:32-34
[22]Hiroshi Shimizu, Taiji Mizuguchi,et. Nisin Production by a Mixed-Culture System Consisting of Lactococcus lactis and Kluyveromyces marxianus[J]. Applied and Environmental Microbiology,1999,65(7):3134-3141
[23]伊首亮.利用浓醪酒糟发酵生产乳酸链球菌素的研究.学位论文.江南大学.2004.
[2] Rogers LA, whiter EO. Limiting Factors in the lactic fermentation [J]. Journal of Bacteriology. 1928,16:211- 229.
[3] Mattick A T R, Hirsch A. A Powerful inhibitory Substance produced by group N Streptococci [J]. Nature(London).1944,154:551.
[4] de Vos WM, Mulders JW, Siezen RJ, et al. Properties ofNisinZ and distribution of its gene,nisZ, in Lactococcus lactis [J ] . Applied and Environmental Microbiology. 59 (1): 213 -218.
[5] Morihiko Meeda, Takashi Iwashita Yoshie Kurihara. Studies on Modifiers. .ⅡPurification and Structure Determination of Grmnemic Acids, Antisweet Active Principle from Grmnema. Sylvestre Leaves[J].Tetrahedron Letters.1989, 30(12):1547-1550.
[6]还连栋,陈秀珠,才迎,等.乳链菌肽产生菌的定向筛选及发酵产物的鉴定[J].微生物学报.1997, 37(4):292-300.
[7]庄绪亮,马桂荣.乳链菌肽的特殊结构与功能[J] .生命的化学.1998, 18 (2):37– 39.
[8]. LLin,PDuearme,EBreukink,etal.ComPutationalstudyofNisininteraetionwithmodel membrane.BioehimBiophysaeta,1999,420(1):111一120
[9].EADavies,HEBevis,JDBroughton.TheuseofthebaeterioeinNisin,asapreservatives inrieott--atyPeeheesestoeontrolthefood一bomePathogenListeriamonoeytogenes.APPI Mierobiol,1997,24:343一346
[10]陈刚新,谢红国,杨丰科.发酵工艺代谢调控[J].化学与生物工程2005, 5:9-11
[11]孔保华,Nisin在红肠保鲜中的应用,酿酒科技,1999(2):38-44.
[12]袁秋萍,乳酸链球菌素在肉制品中的应用, 1998(04):27-28.
[13]汤凤霞,蔡慧农.微生物防腐剂Nisin的研究进展[J].世界农业.2003,(2):43-44.
[14]郭清泉,张兰威,冯镇等.Nisin在酸奶制品中的应用[J].食品与机械.2001,(4):30-31.
[15] Radler. Local measurements of viscoelastic moduli of entangled actin networks using an oscillating magnetic bead micro-rheometer. Biophys J. 1994,66(6): 2210–2216.
[16]庄绪亮,张洪勋,马桂荣,等.乳链菌肽发酵的主要影响因子.化工冶金,2000,21(1):93-97
[17]吴琼,吕红线,王锦,等.乳链菌肽高产菌株发酵条件的研究.工业微生物,2000,30(1):36-39
[18]侯革非,何仁.乳酸链球菌素抑菌效果的研究广西工学院学报,2001,12(4):63-65
[19]庄绪亮.乳链菌肽发酵的主要影响因子[J].化工冶金,2000,21(1):93-97
[20]李孱,白景华.细菌素发酵培养基的优化及动力学初步分析[J].生物工程学报,2001,17(2):187-192
[21]季伟,徐学明.乳酸链球菌固态发酵豆粨的研究[J].饲料资源开发与利用,2006,5:32-34
[22]Hiroshi Shimizu, Taiji Mizuguchi,et. Nisin Production by a Mixed-Culture System Consisting of Lactococcus lactis and Kluyveromyces marxianus[J]. Applied and Environmental Microbiology,1999,65(7):3134-3141
[23]伊首亮.利用浓醪酒糟发酵生产乳酸链球菌素的研究.学位论文.江南大学.2004.
© 2004-2018 中国地质图书馆版权所有 京ICP备05064691号 京公网安备11010802017129号 地址：北京市海淀区学院路29号 邮编：100083 电话：办公室：(+86 10)66554848；文献借阅、咨询服务、科技查新：66554700 |