为了取消ε-聚赖氨酸(ε-poly-L-lysine,ε-PL)提取工艺中的固液分离操作,降低分离提取成本和提高提取效率,该研究建立了一种利用膨胀床直接从发酵液中吸附提取ε-PL的新工艺。首先通过静态吸附进行大孔强酸树脂筛选,再采用两种等温线模型研究293~310 K下树脂吸附ε-PL热力学行为,再对膨胀床吸附提取ε-PL的工艺参数进行优化,最后构建并优化了三柱串联膨胀床吸附提取ε-PL循环工艺条件。结果表明,大孔强酸钠型树脂SQD-04适用于膨胀床工艺,且其吸附ε-PL符合Langmuir吸附等温线模型,热力学参数分析发现SQD-04树脂吸附ε-PL过程为放热反应;在三柱串联膨胀床循环工艺中,初始进料质量浓度25 g/L,树脂初始高径比2.7,膨胀比1.6,v1=1 BV/h时进料,13个柱体积时3#柱穿透,1#柱的树脂饱和度可达0.76,总收率为98.20%。研究结果为利用膨胀床直接从发酵液中吸附提取ε-PL工艺的工业应用提供了重要指导,也为升级换代现有ε-PL提取工艺提供了新思路。
In order to cancel the solid-liquid separation operation in the extraction process of ε-poly-L-lysine(ε-PL), reduce the cost of separation and extraction and improve the extraction efficiency, this study establishes a new process for directly adsorbing and extracting ε-PL from the fermentation broth using expanded bed absorption. Firstly, static adsorption experiments compared and screened the adsorp- tion and desorption parameters of eight macroporous strong acid resins. Secondly, the Langmuir adsorption isotherm model and the Freun- dlich adsorption isotherm model were used to linearly fit the experimental results of the resin adsorption of ε-PL in fermentation broth at 293-310 K, and the experiment studied thermodynamic parameters. Thirdly, single-factor experiments optimized the process parameters (expansion ratio, initial height-diameter ratio of resin, initial feed mass concentration) of ε-PL in the fermentation broth. Finally, the experiments constructed a three-column series expanded bed cycle process and optimized its feed rate. The results showed that the macroporous strong acid resins SQD-04, D072 and D061 were screened out according to the experimental results of the resin working exchange capacity, desorption rate, protein removal rate and pigment removal rate, using HPLC to further analyze the purity and the degree of polymerization of the eluents of the three resins, the SQD-04 resin was screened out to be more suitable for the expanded bed absorption. The correlation coefficient of Langmuir adsorption isotherm model linearly fitting the experimental results of Na+ form SQD-04 resin adsorption of ε-PL in fermentation broth was greater than 0.99. Thermodynamic parameters showed that the adsorption process was an exothermic re-action and the total disorder of the system increases and adsorption of ε-PL in fermentation broth by Na+ form SQD-04 resin was more suit-able at 293 K. In the single factor optimization of expanded bed process parameters, if the column efficiency is considered, the initial feed mass concentration was 25 g/L, the initial height-diameter ratio of the resin was 4.0, the expansion ratio was 1.6; if the process efficiency was considered, the initial feed mass concentration was 25 g/L, the initial height-diameter ratio of the resin was 2.7, the expansion ratio was 1.6. According to the requirements of practical process application, the experiment constructs a three-column series expanded bed circulating process. In the optimization of the three-column series expanded bed cycle process, the initial feed mass concentration was 25 g/L, the initial height-diameter ratio of the resin was 2.7 and the expansion ratio was 1.6 as fixed process parameters, the results show that when v1=1 BV/h, the 3# column penetrates at 13 column volumes, the resin saturation of the 1# column can reach 0.76, and the total yield was 98.20%. These research results prove that the three-column series expanded bed circulation process can improve the extraction efficiency, provide important guidance for the industrial application of the ε-PL process directly from the fermentation broth using the expanded bed adsorption and a new idea for upgrading the existing ε-PL extraction process, but if the process is to be scaled up and used in industry, it should be combined with a simulated moving bed design in order to realize continuous adsorption-washing-elution operation.
[1] 赵星宇, 陈旭升, 张宏建, 等.膜分离工艺提高产品中高聚合度ε-聚赖氨酸含量[J].食品与发酵工业, 2021, 47(14):196-201.
ZHAO X Y, CHEN X S, ZHANG H J, et al.Increasing the ε-PL content with high degree of polymerization in products using membrane separation[J].Food and Fermentation Industries, 2021, 47(14):196-201.
[2] 齐子琦, 秦子晋, 黄永震, 等.生物防腐剂ε-聚赖氨酸的研究进展[J].食品工业, 2019, 40(10):289-293.
QI Z Q, QIN Z J, HUANG Y Z, et al.Research progress of ε-polylysine as a biological preservative[J].The Food Industry, 2019, 40(10):289-293.
[3] 邹凯华, 展海军.ε-聚赖氨酸作为食品防腐剂的应用[J].食品研究与开发, 2008, 29(1):165-168.
ZOU K H, ZHAN H J. Application of ε-poly-L-lysine as food preservative[J].Food Research and Development, 2008, 29(1):165-168.
[4] ZHOU Y P, YAN P, TANG L.Self-protection of Streptomyces to ε- poly-L-lysine improves fermentation efficacy[J].Biochemical Engineering Journal, 2021, 168:107935.
[5] WANG L, LI S, ZHAO J J, et al.Efficiently activated ε-poly-L-lysine production by multiple antibiotic-resistance mutations and acidic pH shock optimization in Streptomyces albulus[J].Microbiologyopen, 2019, 8(5):e00728.
[6] SHIMA S, SAKAI H.Poly-L-lysine produced by Streptomyces.part II.taxonomy and fermentation studies[J].Agricultural and Biological Chemistry, 1981, 45(11):2 497-2 502.
[7] US Food and Drug Administration.GRN 000135-2004b ε-poly-lysine for addition to specified foods[S].Washington D.C.U.S.Office of Food additive Safty, 2005.
[8] CHEN X S, GAO Y, ZHEN B, et al.Separation and purification of ε-poly-L-lysine from fermentation broth[J].Process Biochemistry, 2016, 51:134-141.
[9] CHEN X S, WANG K F, ZHENG G C, et al.Preparation, characterization and antimicrobial activity of ε-poly-L-lysine with short chain length produced from glycerol by Streptomyces albulus [J].Process Biochemistry, 2018, 68:22-29.
[10] DE SOUSA F C Jr, DE ARAU'JO PADILHA C E, CHIBÉRIO A S, et al.Modeling and simulation of breakthrough curves of recombinant 503 antigen using immobilized metal affinity expanded bed adsorption chromatography[J].Separation & Purification Technology, 2016, 164:34-40.
[11] PATHAPATI T, RUTZE D N, DE WIT P, et al.Innovation of expanded bed adsorption by integrating simulated moving bed technology[J].Chemical Engineering & Technology, 2018, 41(12):2 393-2 401.
[12] MACIEL K S, SANTOS L S, BONOMO R C F, et al.Purification of lactoferrin from sweet whey using ultrafiltration followed by expanded bed chromatography[J].Separation and Purification Technology, 2020, 251:117324.
[13] 郭静, 王峰, 崔正刚, 等.膨胀床-固定床层析偶联方式分离纯化藻蓝蛋白[J].食品科学, 2013, 34(10):107-111.
GUO J, WANG F, CUI Z G, et al.Separation and purification of phycocyanin by combined use of expanded bed and packed bed[J].Food Science, 2013, 34(10):107-111.
[14] ITZHAKI R F.Colorimetric method for estimating polylysine and polyarginine[J].Analytical Biochemistry, 1972, 50(2):569-574.
[15] BRADFORD M M. A repid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[J].Analytical Biochemistry, 1976, 72(1):248-254.
[16] ZHEN B, CHEN X S, HAN D, et al.An alternative method for the decoloration of ε-poly-L-lysine eluate by microporous resin in the separation and purification of ε-poly-L-lysine from fermentation broth[J].Food and Bioproducts Processing, 2015, 95:332-338.
[17] XU D L, WANG R, XU Z X, et al.Discovery of a short-chain ε- poly-L-lysine and its highly efficient production via synthetase swap strategy[J].Journal of Agricultural and Food Chemistry, 2019, 67(5):1 453-1 462.
[18] 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.
FAN B Q, JIA S R, DAI Y J, et al.The quality analysis of ε-poly- L-lysine produced by fermentation[J].China Food Additives, 2010(3):108-112.
[19] FINETTE G M S, MAO Q M, HEARN M T W.Comparative studies on the isothermal characteristics of proteins adsorbed under batch equi- librium conditions to ion-exchange, immobilised metal ion affinity and dye affinity matrices with different ionic strength and temperature con- ditions[J].Journal of Chromatography A, 1997, 763(1):71-90.
[20] 高阳, 何洪刚, 李芳良, 等.Amberlite IRC-50树脂吸附与解吸ε-聚赖氨酸的过程优化[J].过程工程学报, 2016, 16(4):601-606.
GAO Y, HE H G, LI F L, et al.Optimization and evaluation on adsorption and desorption of ε-poly-L-lysine on amberlite IRC-50 resins[J].The Chinese Journal of Process Engineering, 2016, 16(4):601-606.
[21] 何洪刚, 李芳良, 马玉, 等.钠型Amberlite IRC-50树脂分离提取ε-聚赖氨酸的条件优化[J].食品与发酵工业, 2018, 44(7):148-153;160.
HE H G, LI F L, MA Y, et al.Optimization of conditions for sepa- ration and extraction of ε-poly-L-lysine from fermentation broth by Na+ form Amberlite IRC-50 resin[J].Food and Fermentation In-dustries, 2018, 44(7):148-153;160.
[22] KITO M, TAKIMOTO R, YOSHIDA T, et al.Purification and characterization of an ε-poly-L-lysine-degrading enzyme from an ε- poly-L-lysine-producing strain of Streptomyces albulus[J].Archives of Microbiology, 2002, 178(5):325-330.
[23] HAMANO Y, YOSHIDA T, KITO M, et al.Biological function of the pld gene product that degrades-poly-L-lysine in Streptomyces albulus[J].Applied Microbiology and Biotechnology, 2006, 72(1):173-181.
[24] FIROUZTALE E, MAIKNER J J, DEISSLER K C, et al.Valida-tion of a theoretical model for adsorption using cephalosporin C and polymeric reversed-phase resins[J].Journal of Chromatography A, 1994, 658(2):361-370.
[25] LIKOZAR B, SENICA D, PAVKO A. Interpretation of experimental results for vancomycin adsorption on polymeric resins in a fixed bed column by mathematical modeling with independently estimated parameters[J].Industrial & Engineering Chemistry Research, 2013, 52(26):9 247-9 258.
