NaCS-PDMDAAC微胶囊固定化红曲霉菌培养的研究
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摘要
首先采用平板初筛—摇瓶复筛的筛选策略，从原始菌株筛选得到一株液态发酵下高产红曲色素的红曲霉菌菌株Monascus purpureus，摇瓶发酵时该菌株红曲色素产量为1.59(OD_(500))。然后针对Monascus sp.液态发酵的外部条件如供氧量和温度进行优化，确定最佳游离发酵条件，最后对发酵培养基的初始pH值、碳源和氮源进行进一步优化获得最佳的液体培养基组成，摇瓶发酵的红曲色素产量从优化前的1.59(OD_(500))提高到1.93(OD_(500))。
采用纤维素硫酸钠(NaCS)—聚二甲基二烯丙基氯化铵(PDMDAAC)微胶囊对红曲霉菌进行了固定化培养试验。由于培养环境的变化，考察了红曲霉菌微囊化对摇瓶培养条件的影响，然后在该优化条件下连续进行6批培养。结果表明，红曲霉菌可以很好地在NaCS-PDMDAAC微胶囊中生长和产红曲色素。在与游离培养细胞的比较表明，微囊化培养的糖耗时间会远远少于游离培养，红曲色素的浓度在前3批略低于游离培养，但一旦达到稳定后，就会高于游离培养，最高红曲色素产量为2.49(OD_(500))。同时发现，微囊化培养时红曲霉菌的菌体浓度远大于游离培养，达到13.4g·L~(-1)。
设计了一个高25cm，低面半径为3.5cm的鼓泡塔反应器，在与传统发酵罐和摇瓶的对比中，鼓泡塔反应器减少剪切应力对胶囊的破坏，同时增加了氧的供应能力，适合微囊化红曲霉菌的培养。
对微囊化红曲霉菌的鼓泡塔反应器培养条件进行优化，选择最佳的初始糖浓、胶囊装载量和通气速率。在连续十五批的分批培养中，第一批发酵时，囊内的菌体不断生长，并没有受到微胶囊的限制，因此相比前面游离培养和微囊化摇瓶培养，葡萄糖消耗最快，红曲色素产量也为最高。随着一批又一批发酵的进行，除了囊内菌浓继续增加外，发酵周期也不断缩短，稳定后的红曲色素产量已经达到游离培养时的2倍，而生物量达到游离培养的3倍。这说明鼓泡塔反应器能够满足足够多生物量的红曲霉菌对氧的需求，最后红曲色素的平均产率为0.158(OD_(500)·h~(-1))，为游离培养时产率的10倍左右。
Cultivation of Monascus purpurpeus for production of natural pigments was investigated. A strain of Monascus with high-throughput of pigments in submerged culture named Monascus sp. was obtained through two-step screening strategy, plate selecting and shaking culture determining. The yield of red pigment by Monascus sp. obtained in liquid culture was 1.59 (OD500)
The fermentation conditions were optimized to gain the maximum yield of pigments in submerged culture of Monascus sp.. In the first optimization step, the influence of external factors, such as dissolved oxygen and temperature, on pigment production was evaluated and the optimal external conditions were determined. In the second step, the concentrations of initial pH and initial concentrations of glucose, NaNO3 and yeast extract were further optimized to determine the ingredients of the medium. The optimized conditions allowed the pigment production to be increased from 1.59 to 1.93 (OD500) A bubble column, with the diameter and height of 3.5 cm and 25 cm respectively, was made from glass. In comparison with flasks and conventional bioreactor, the bubble column was applied to culture encapsulated Monascus sp. due to its better oxygen transfer property and less stress on microcapsules.
Production of pigment in submerged culture was much less than that in solid-state fermentation, since the solid-state culture provided a support for the mycelium. The fermentation of Monascus sp. immobilized in polyelectrolyte complex (PEC) microcapsules, which were prepared by sodium cellulose sulphate and poly-dimethyldiallylammonium chloride, was a good substitute for submerged culture because it mimicked the solid-state environment. The repeated-batch process with encapsulated cells was studied in flasks and a bubble column. It was found that the bubble column was more suitable for encapsulation culture than conventional bioreactor and flasks because of its excellent mass transfer properties and minor breaking stress on microcapsules. Owing to the protection of the microcapsules, the biomass in microcapsules increased to approximately 3 times that in free culture with negligible cell leakage to the medium. And the pigment yield in the bubble column finally reached 3.82 (OD500) which was 2-fold augment, while the period of each batch was shortened to 15% of that in free culture. In the repeated-batch process, the mean productivity of pigment by encapsulated Monascus sp. was 0.158(OD500 h-1), which was nearly 10 times that by free cells. In the kinetic analysis of metabolite production, some changes in pigment production were observed in the repeated-batch process.
采用纤维素硫酸钠(NaCS)—聚二甲基二烯丙基氯化铵(PDMDAAC)微胶囊对红曲霉菌进行了固定化培养试验。由于培养环境的变化，考察了红曲霉菌微囊化对摇瓶培养条件的影响，然后在该优化条件下连续进行6批培养。结果表明，红曲霉菌可以很好地在NaCS-PDMDAAC微胶囊中生长和产红曲色素。在与游离培养细胞的比较表明，微囊化培养的糖耗时间会远远少于游离培养，红曲色素的浓度在前3批略低于游离培养，但一旦达到稳定后，就会高于游离培养，最高红曲色素产量为2.49(OD_(500))。同时发现，微囊化培养时红曲霉菌的菌体浓度远大于游离培养，达到13.4g·L~(-1)。
设计了一个高25cm，低面半径为3.5cm的鼓泡塔反应器，在与传统发酵罐和摇瓶的对比中，鼓泡塔反应器减少剪切应力对胶囊的破坏，同时增加了氧的供应能力，适合微囊化红曲霉菌的培养。
对微囊化红曲霉菌的鼓泡塔反应器培养条件进行优化，选择最佳的初始糖浓、胶囊装载量和通气速率。在连续十五批的分批培养中，第一批发酵时，囊内的菌体不断生长，并没有受到微胶囊的限制，因此相比前面游离培养和微囊化摇瓶培养，葡萄糖消耗最快，红曲色素产量也为最高。随着一批又一批发酵的进行，除了囊内菌浓继续增加外，发酵周期也不断缩短，稳定后的红曲色素产量已经达到游离培养时的2倍，而生物量达到游离培养的3倍。这说明鼓泡塔反应器能够满足足够多生物量的红曲霉菌对氧的需求，最后红曲色素的平均产率为0.158(OD_(500)·h~(-1))，为游离培养时产率的10倍左右。
Cultivation of Monascus purpurpeus for production of natural pigments was investigated. A strain of Monascus with high-throughput of pigments in submerged culture named Monascus sp. was obtained through two-step screening strategy, plate selecting and shaking culture determining. The yield of red pigment by Monascus sp. obtained in liquid culture was 1.59 (OD500)
The fermentation conditions were optimized to gain the maximum yield of pigments in submerged culture of Monascus sp.. In the first optimization step, the influence of external factors, such as dissolved oxygen and temperature, on pigment production was evaluated and the optimal external conditions were determined. In the second step, the concentrations of initial pH and initial concentrations of glucose, NaNO3 and yeast extract were further optimized to determine the ingredients of the medium. The optimized conditions allowed the pigment production to be increased from 1.59 to 1.93 (OD500) A bubble column, with the diameter and height of 3.5 cm and 25 cm respectively, was made from glass. In comparison with flasks and conventional bioreactor, the bubble column was applied to culture encapsulated Monascus sp. due to its better oxygen transfer property and less stress on microcapsules.
Production of pigment in submerged culture was much less than that in solid-state fermentation, since the solid-state culture provided a support for the mycelium. The fermentation of Monascus sp. immobilized in polyelectrolyte complex (PEC) microcapsules, which were prepared by sodium cellulose sulphate and poly-dimethyldiallylammonium chloride, was a good substitute for submerged culture because it mimicked the solid-state environment. The repeated-batch process with encapsulated cells was studied in flasks and a bubble column. It was found that the bubble column was more suitable for encapsulation culture than conventional bioreactor and flasks because of its excellent mass transfer properties and minor breaking stress on microcapsules. Owing to the protection of the microcapsules, the biomass in microcapsules increased to approximately 3 times that in free culture with negligible cell leakage to the medium. And the pigment yield in the bubble column finally reached 3.82 (OD500) which was 2-fold augment, while the period of each batch was shortened to 15% of that in free culture. In the repeated-batch process, the mean productivity of pigment by encapsulated Monascus sp. was 0.158(OD500 h-1), which was nearly 10 times that by free cells. In the kinetic analysis of metabolite production, some changes in pigment production were observed in the repeated-batch process.
引文
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[2]. Klerin, J., Wagner, F., Methods for the immobilization of microbial cells, Applied Biochemistry and Bioengineering, Vol. 4, New York: Academic Press, 1983
[3]． 梅乐和，姚善泾，生物微胶囊固定化技术的研究进展，现代化工，1998，1：19-21
[4]. Bailey, J. E., Ollis, D. F., Biochemical Engineering Fundamentals, New York: McGraw Hill, 1997
[5]. Westrin, A., Axelson, A., Diffusion in gels containing immobilized cells-A critical review, Biotechnology Bioengineering, 1991,38:439-443
[6]. Cosnier, S., Biomolecule immobilization on electrode surfaces by entrapment or attachment to electrochemically polymerized films: A review, Biosensors and Bioelectronics, 1999,14:443-456
[7]． 居乃琥，固定化细胞技术研究和应用的现状与展望(一)，工业微生物，1989，19(1)：25-32
[8]． 居乃琥，固定化细胞技术研究和应用的现状与展望(二)，工业微生物，1989，19(2)：25-33
[9]． 梁治中，微胶囊技术及其应用，北京：中国轻工业出版社，1999
[10]. Chang, T. M. S., Semipermeable microcapsules, Science, 1964, 146:524-525
[11]. Chang, T. M. S., Semipermeable microcapsules containing catalase for enzyme replacement in acatalasemic mice, Nature, 1968, 218:243-244
[12]. Park, J. K., Chang, H. N., Microencapsulation of microbial cells, Biotechnology Advance, 2000, 18:303-319
[13]. Jensen, V. J., Rugh, S., Industrial-scale production and application of immobilized glucose isomerase, Method in Enzymology, 1987, 136:356-359
[14]． 绀户朝治著，阎世翔译，微胶囊化工工艺学，轻工业出版社，1989
[15]． 宁国伯，郭明珠，微胶囊形成的理化条件和影响因素，生物工程学报，1988，4(3)：226-229
[16]． 虞星炬，解玉冰，马小军，袁权，第七届全国生物化工学术会议论文集，北京：化学工业出版社，1996：11-15
[17]. Prakash, S., Chang T. M. S., Preparation and in vitro analysis of microencapsulated genetically engineered E. coli DH5 cells for urea and ammonia removal, Biotechnology & Bioengineering, 1995, 46:621-626
[18]. Lim, F., Sun, A. M., Microencapsulated islets as bioartificial endocrine pancrease, Science, 1980, 210:908-910
[19]. Douglas, B. S., Janice, A. P., Porous alginate-poly(ethylene glycol) entrapment system for the cuttiration of mammalian cells, Biotechnology Progress, 1997,
13:569-576
[20]. King, G. A., Daugulis A. J., Alginate-polylysine microcapsules of controlled membrane molecular weight cutoff for mammalian cell culture engineering, Biotechnology Progress, 1987, 3:231-240
[21]. Wang, F. F., Wu C. R., Wang, Y. J., Preparation and application of poly(binylamine)/alginate microcapsules to culturing of a mouse erythroleukemia cell line, Biotechnology & Bioengineering 1992, 40:1115-1118
[22]. Yoshioka, T., Hirano, R., Shioya, T., Encapsulation of mammalian cell with chitosan-CMC capsule, Biotechnology & Bioengineering, 1990, 35:66-72
[23]. Jin, Y. B., Park J. K., Recovery of lead using encapsulated Saccharomyces cerevisiae, JKIChE, 1998, 36:229-234
[24]. Oh, C. Y., Park, J. K., The characteristics of encapsulated whole cell β-galactosidase, Bioprocess Engineering, 1998, 19:419-425
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