乳酸菌细菌素发酵及分离纯化工艺优化
文献类型：学位论文
| 作者 | 李孱 |
| 学位类别 | 博士 |
| 答辩日期 | 2000-10 |
| 授予单位 | 中国科学院研究生院 |
| 导师 | 欧阳藩 |
| 关键词 | 细菌素发酵 双水相萃取 纯化 响应面方法 优化 |
| 其他题名 | Qptimization of conditions for fermentation and extraction and purification of bacteriocin produced by lactoccus ladtis |
| 学位专业 | 生化工程 |
| 中文摘要 | 提高细菌素产率、简化分离纯化工艺、降低生产成本是天然食品防腐剂研究与开发面临的难题。为此，本文以乳链菌肽为对象，针对目前细菌发酵及分离纯化存在的关键问题，利用生化工程的新技术进行了双水相萃取细菌素发酵规律；细菌素在双水相中的分配规律；双水相及温度诱导双水相萃取分离纯化细菌素；响应面方法优化培养基以及菌体生长动力学的探索和研究。为细菌素发酵条件的优化、发酵与产物分离的耦合及细菌素的分离纯化提供了新的途径和思路。为了解除发酵过程副产物对菌体生长和目标产物积累的抑制，本文首次提出在PEG/盐双水相系统中萃取培养耐盐微生物。对于双水相萃取L.lactis ATCC 11454产生的乳链菌肽发酵，菌株生长和目的产物积累最适的双水相系统是11%PEG20000/3.5% MgSO_4·7H_2O。菌株ATCC 11545在该系统中完全分配在下相，在同样的原培养基组份下，其生物量可达单相（对照）培养的60%，而产物产量则可提高33%。双水相系统系线长度和相体积比的改变均变均影响菌株的生长和目的产物的积累。PEG/盐双水相系统萃取发酵可延伸到耐高渗微生物的培养中以解除产物抑制和简化产物分离工艺。论文发表于Biotechnology Letters上。研究了细菌素在PEG/盐双水相系统中的分配规律。相系统的组成影响着细菌素在双水相系统中的分配，PEG分子量越小越有利于细菌素萃取到PEG相中；PEG浓度越大，系统的疏水性差异越强，越有利于细菌素在PEG相的分配；中性盐Na_2SO_4有利于细菌素的萃取，盐浓度越大，盐析能力增大而有利于细菌素分配系数的提高。同时，pH值也影响着细菌素在双水相系统中的分配，在酸性区域，细菌素在pH2时的分配系数较大，在碱性区域随着pH值的增加分配系数增大，这是由于除了疏水作用外，还有静电作用使得细菌素在碱性环境中具有较大的分配系数，但在碱性区域细菌素有活性的损失。细菌素在双水相系统中的分配还受表面活性剂的强烈影响，阳离子表面活性剂能有效地使细菌素分配于下相：阴离子和两性表面活性剂则相反。导致乳链菌肽在双水相系统中分配的作用力主要是疏水作用力，这有别于普通蛋白质在双水相中的分配。在了解细菌素在双水相系统中分配规律的基础上，用响应面方法优化了PEG/盐双水相系统萃取细菌素标样的工艺条件。双水相萃取细菌素标样的最佳系统组成为：14.54% PEG 4000/12.47% Na_2SO_4 + 1% TritonX-100 (pH2),在此条件下双水相系统萃取细菌素标样的模型预测和实测收率可达100%，但在此条件下萃取细菌素发酵液，其收率只有90%。细菌素发酵液的萃取条件，尚须进行必要的优化。首次用统计实验设计的方法进行了PEG/盐双水相系统萃取细菌素发酵液的工艺条件的优化，得出在pH 2条件下成相组份PEG 4000和Na_2SO_4的最佳浓度分别为：15.99% (w/w) PEG 4000和15.85% (w/w) Na_2SO_4 (pH 2)。在此条件下，双水相系统萃取细菌素发酵液的回收量比用标准乳链菌肽检测的原发酵液的细菌素高出11.6%。证明PEG/盐双水相系统是萃取分离细菌素的一种快速而效的方法，它集成了产物萃取与细胞的去除。本文还利用TritonX-114温度诱导双水相系统能萃取疏水性蛋白的性质，研究了细菌素的分离纯化。实验证明此方法简便快速，能将细菌素有效地富集到疏水相中。成相成份和离子强度是影响细菌素分离纯化的主要因素。优化得出的最适分离纯化条件为：3.27% TritonX-114和0.85 M的氯化钠，在此条件下乳链菌肽萃取率为92%，纯化系数为9.05。从提高目的产物产量及有利于下游产物分离的角度出发，首次用响应面方法对目前细菌素发酵产量较高的培养基进行了进一步优化，得到了组成为(gL~(-1))：蔗糖，10；大豆蛋白胨，4.49；酵母粉，10；KH_2PO_4，28.42；NaCl，2；MgSO_4·7H_2O，0.2的优化培养基，不仅有机氮源大为减少，有利于后续的分离纯化，而且细菌素产量提高一倍。菌株在优化前后培养基中的发酵动力学性质相近，与菌体均为部分耦联型。研究结果表明响应面方法在细菌素发酵培养基优化及双水相萃取分离纯化工艺条件的优化中是非常有效的工具。它能快速地找出主效及最佳操作工艺条件，使得目的响应具有最大值。 |
| 英文摘要 | The low productivity, the complicated technology of isolation and purification, and the high production cost of bacteriocins are the bottleneck for the research and development of natural food preservatives. In order to solve these vital problems, the followings were investigated using nisin as a representative of bacteriocins, including (1) extractative fermentation of a bacteriocin in aqueous two-phase systems (ATPS); (2) partitioning rule of nisin in PEG/salt ATPS; (3) extracting the bacteriocin from the broth using PEG/salt ATPS; (4) isolating and purifying the bacteriocin from the broth using temperature-induced ATPS based on detergent of TritonX-114; (5) optimizing a medium of bacteriocin fermentation using a response surface methodology; and (6) growth kinetics for L. lactis ATCC 11454. The findings of this work could provide sound basis for optimization of conditions for bacteriocin fermentation, integration of bacteriocin fermentation and extraction, and isolation and purification of bacteriocins. To relieve the inhibition of the by-product produced in the bacteriocin fermentation to cell growth and accumulation of the desired product, it was first proposed that the salt-tolerant microbes be cultivated in PEG/salt aqueous two-phase systems. The optimum component of ATPS for the cell growth of L. lactis ATCC 11454 and nisin production was 11% PEG 20000/3.5% MgSO_4·7H_2O. In this ATPS medium, the cells were completely partitioned in the bottom phase, and the cell biomass was only 60% of the control medium, but bacteriocin production was enhanced by 33%. The changes of tie-line length and phase volume ratio for the identical tie line was able to affect cell growth and bacteriocin accumulation. Extractive fermentation in PEG/salt aqueous two-phase systems may be extended to the microbes with the ability to tolerate high osmotic pressure. By the way of extractive fermentation in PEG/salt ATPS, the product inhibition can be relieved and the processes of isolation and purification of the desired product can be simplified. The partitioning rule of bacteriocin in PEG/salt aqueous two-phase systems was first studied. The partitioning coefficient of bacteriocin in ATPS was affected by the constituent, pH, and surftactants of the ATPS. The constituent of ATPS influenced the partitioning of bacteriocin in aqueous two-phase systems. The smaller the molecular weight of PEGs was, the larger the partitioning coefficient for bacteriocin was. The hydrophobic difference between the two phases in an ATPS, which was responsible for bacteriocin extraction, was increased with the elevated PEG concentration. A neutral salt, sodium sulfate, allowed bacteriocin in the ATPS to be of the biggest partitioning coefficient compared to acidic and alkaline salts. The partitioning coefficient of bacteriocin was enhanced with the salt concentration increased and the salt-out capability augmented. The effects of pH on the bacteriocin partition in ATPS were complicated. The pH value of 2 allowed bacteriocin in the ATPS to have a bigger partitioning coefficient when pH was in the range from 2 to 6. The partitioning coefficient of bacteriocin in ATPS was increased with the elevated pH value when pH was greater than 5, but there were some loss of bacteriocin activities in alkaline areas. Also, surfactants strongly influenced the bacteriocin partitioning. Cation surfactants were effectively able to make bacteriocin be partitioned to the bottom phase of ATPS, and anion and amphiphilic surfactants were able to make bacteriocin be partitioned to the top phase of ATPS. The main action strength that made bacteriocin have different partition in ATPS was hydrophobic action, and this was different from most of proteins to be partitioned in ATPS. Based on the partitioning rule of bacteriocin in ATPS, the conditions of extracting the standard bacteriocin sample in PEG/salt ATPS were optimized using a response surface methodology. The optimum constituent of PEG/salt ATPS for extracting bacteriocin standard was 14.54% PEG 4000 / 12.47% Na_2SO_4 + 1% TritonX-100 (pH2). The model for extracting the standard bacteriocin sample predicted bacteriocin to be completely extracted to the top phase of ATPS under the optimum conditions, and the observed recovery in experiments confirmed these results, but the recovery of bacteriocin for extracting bacteriocin from the broth under the same optimum conditions was only 90%. Therefore, the conditions for extracting bacteriocin from the broth had to be optimized further. The conditions for extracting bacteriocin from the broth using PGC/salt ATPS were also first optimized using a response surface methodology. The optimum constituent of PEG/salt ATPS for extracting bacteriocin from the broth was 15.99% (w/w) PEG 4000/15.85 (w/w) Na_2SO_4 (pH 2). Under the optimum conditions, the bacteriocin yield was increased by 11.60% compared to that assayed in the same broth by the standard method of nisin assay. The findings showed that it was a rapid and effective method for PEG/salt aqueous two-phase systems to extract bacteriocin from the broth because of the high recovery and integration of cell removal. Isolation and purification of bacteriocin was also studied in this article using the property that temperature-induced aqueous two-phase systems based on TritonX-114 can extract hydrophobic proteins. The experimental results indicated that this method was a rapid and simplified one and could effectively concentrated the bacteriocin to the hydrophobic phase. The concentration of detergent TritonX-114 and the ion strength were the two main factors that affected the bacteriocin partitioning, and the optimum conditions were 3.27% TritonX-114, and 0.85 M NaCl and allowed the temperature-induced ATPS to get 92% recovery and 9.05 purification factor. In order to enhance the productivity of bacteriocin and simplify the purification procedure of the desired product, the medium, which allows the bacteriocin-producer to have high yield of bacteriocin and is now widely used by researchers, was first optimized using a response surface methodology. The optimum medium (gL~(-1)) was: 10 sucrose, 4.49 soybean peptone, 10 yeast extract, 28.42 KH_2PO_4, 2NaCl, 0.2 MgSO_4·7H_2O. This optimization decreased organic nitrogen source, made the bacteriocin be isolated and purified easily, and allowed the bacteriocin yield to be double. Also, the kinetics of bacteriocin fermentation for L. latis ATCC 1145 in the media before and after the optimization were compared and It was found that the kinetics were similar and partially growth-associated. The research results from the thesis showed that the response surface methodology was proved to be a powerful and useful tool for optimizing the conditions of fermentation and purification for bacteriocin. It can help the researchers find the main effects rapidly and the optimum conditions that made the desired response to have the optimum level. |
| 语种 | 中文 |
| 公开日期 | 2013-09-26 |
| 页码 | 153 |
| 源URL | [http://ir.ipe.ac.cn/handle/122111/1921]  |
| 专题 | 过程工程研究所_研究所（批量导入） |
| 推荐引用方式 GB/T 7714 | 李孱. 乳酸菌细菌素发酵及分离纯化工艺优化[D]. 中国科学院研究生院. 2000. |
入库方式： OAI收割
来源：过程工程研究所
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