产阿扎霉素F菌株Streptomyces malaysiensis种子培养条件研究
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摘要
【目的】以阿扎霉素产生菌马来西亚链霉菌ECO-00002为试验菌株,优化种子培养基成份和培养条件,提高菌体的生物量。【方法】采用Plackett-Burman试验筛选适宜的碳氮源成分,并通过Response-Surface-Analysis(RSA)试验,找出其最大响应值;进一步通过对比试验,确定复合维生素、复合盐、CaCO_3的配比,同时优化pH值、装液量等种子培养条件。【结果】菌株种子生长主要的影响碳氮源为葡萄糖、酵母膏、麦芽膏,其最大响应值分别为1.45%、1.50%和2.0%;无机盐为CaCO_3 3.00 g/L、MgSO_4·7H_2O 1.00 g/L、MnSO_4·4H_2O 0.10 g/L、KH_2PO_4 0.20 g/L;培养条件为初始pH 7.50,装液量100 mL/500 mL,培养温度28℃,培养时间为200 r/min振荡培养40 h。【结论】在优化的种子培养基和培养条件下,菌株菌体生物量由原来的4.09 g提高到8.91 g,提高率为117.70%,优化效果显著。
【Objective】 In order to increase the biomass of the strain S. malaysiensis ECO-00002, the seed medium composition and culture conditions were optimized. 【Method】The Plackett-Burman test was used to screen the carbon and nitrogen sources for seed culture medium, and the Response Surface Analysis test was used to identify the most significant factors and to determine their optimal values. Furthermore, the proportion of multivitamins, complex salts, and CaCO_3 were determined. The initial pH of the medium and liquid volume were optimized by comparative tests. 【Result】 The optimization results showed that the growth of the strain seed mainly affected by the carbon source which were glucose, yeast extract and malt extract, and the maximum response values of the three factors were 1.45 %, 1.50 %, 2.00 %, respectively. The inorganic salt was as follows: CaCO_3 3.00 g/L, MgSO_4·7 H_2O 1.00 g/L, MnSO_4·4 H_2O 0.10 g/L, KH_2PO_4 0.20 g/L. Initial pH was 7.50, and liquid volume was 100 mL/500 mL. The optimization of culture conditions was as following: culture temperature was 28 ℃, and incubation time was 200 r/min with shaking culture 40 hours. 【Conclusion】 Under the optimized seed culture conditions, the biomass yield increased from 4.09 g to 8.91 g, and the increase rate was 117.70 %. The optimization effect was significant.
【Objective】 In order to increase the biomass of the strain S. malaysiensis ECO-00002, the seed medium composition and culture conditions were optimized. 【Method】The Plackett-Burman test was used to screen the carbon and nitrogen sources for seed culture medium, and the Response Surface Analysis test was used to identify the most significant factors and to determine their optimal values. Furthermore, the proportion of multivitamins, complex salts, and CaCO_3 were determined. The initial pH of the medium and liquid volume were optimized by comparative tests. 【Result】 The optimization results showed that the growth of the strain seed mainly affected by the carbon source which were glucose, yeast extract and malt extract, and the maximum response values of the three factors were 1.45 %, 1.50 %, 2.00 %, respectively. The inorganic salt was as follows: CaCO_3 3.00 g/L, MgSO_4·7 H_2O 1.00 g/L, MnSO_4·4 H_2O 0.10 g/L, KH_2PO_4 0.20 g/L. Initial pH was 7.50, and liquid volume was 100 mL/500 mL. The optimization of culture conditions was as following: culture temperature was 28 ℃, and incubation time was 200 r/min with shaking culture 40 hours. 【Conclusion】 Under the optimized seed culture conditions, the biomass yield increased from 4.09 g to 8.91 g, and the increase rate was 117.70 %. The optimization effect was significant.
引文
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[15]戴蓬博,蓝星杰,宗兆锋,等.极长链霉菌sl01菌株抑菌活性物质的发酵条件优化和稳定性研究[J]. 农药学学报, 2016, 18(6): 729-737.
[16]卢冰霞,何颖,赵武,等.应用正交试验优化猪细小病毒N株在IBRS-2细胞转瓶培养[J].西南农业学报, 2017, 30(2):839-842.
[17]邓志平,陈浩,陈三凤.利用响应面法优化固氮类芽孢杆菌paenibacillus sp.1-49的发酵培养基[J]. 微生物学报, 2016, 56(9): 1415-1425.
[18]Liu R S, Tang Y J. Tuber melanosporum fermentation medium optimization by Plackett-Burman design coupled with Draper-Lin small composite design and desirability function[J]. Bioresource Technology, 2010, 101(9): 3139-3146.
[19]洪亮,杨丽芬,杨建,等.小枣黑疔病拮抗放线菌的筛选与发酵优化[J].西南农业学报,2018,31(8):1634-1637.
[20]Sunitha K, Kim Y O, Lee J K, et al. Statistical optimization of seed and induction conditions to enhance phytase production by recombinant escherichia coli[J]. Biochemical Engineering Journal, 2000, 5(1): 51-56.
[21]黄艳,赵春田,裘娟萍.一株加纳链霉菌种子培养工艺及移种标准的研究[J]. 中国畜牧杂志, 2014, 50(7): 79-84.
[22]赵鑫,肖玉平,廖延智,等. L-精氨酸高产菌株的亚硝基胍诱变选育和种子培养基的优化研究[J].中国酿造, 2014, 33(9): 81-85.
[2]冯云,任路静,瞿亮,等.种子培养基成分对裂殖壶菌发酵产DHA的影响[J]. 生物加工过程, 2013, 11(5): 26-31.
[3]Jalil R, Kalil M. S, Rahman N A, et al. Seed culture conditions for high yield of cellulolytic enzymes production from pycnoporus sanguineus[J]. Malaysian Journal of Analytical Sciences, 2017, 21(1): 231- 239.
[4]刘金国,涂璇,涂晓嵘,等.种子工艺优化对链霉菌702发酵产抑真菌生物活性物质的影响[J].江西科学, 2007, 25(3): 253-257.
[5]Khattab A I, Babiker E H, Saeed H A. Streptomyces: isolation, optimization of culture conditions and extraction of secondary metabolites[J]. International Current Pharmaceutical Journal, 2016, 5(3): 27-32.
[6]Elazazy M S, El-Hamshary M, Sakr M, et al. Plackett-burman and box-behnken designs as chemometric tools for micro-determination of l-ornithine[J]. Spectrochimica Acta Part A Molecular & Biomolecular Spectroscopy, 2018, 193:397-406.
[7]Roussouly N, Petitjean F, Salaun M. A new adaptive response surface method for reliability analysis[J]. Probabilistic Engineering Mechanics, 2013, 32: 103-115.
[8]张帅,刘飞,朱华伟,等. Plackett-Burman 设计和响应面法优化单宁酶固态发酵培养基[J].广东农业科学, 2015, 42(12): 91-96.
[9]陈义光,刘祝祥,李铭刚,等.产环己酰亚胺新菌株yim41004种子培养基优化研究初报[J].云南大学学报:自然科学版, 2006, 28(2): 173-177.
[10]Chen G, Wang G Y, Li X, et al. Enhanced production of microbial metabolites in the presence of dimethyl sulfoxide[J]. Journal of Antibiotics, 2000, 53(10): 1145-1153.
[11]陈坚.发酵过程优化原理与实践[M].北京:化学工业出版社, 2002.
[12]ZHANG N, XU D, YIN T, et al. Optimization on Fermentation Condition of Surfactin by Bacillus subtilis ATCC 21332[J]. Journal of Anhui Agricultural Sciences, 2017, 15: 37.
[13]Zhou Y, Lu Z, Wang X, et al. Genetic engineering modification and fermentation optimization for extracellular production of recombinant proteins using escherichia coli[J]. Applied Microbiology & Biotechnology, 2018, 102(4): 1-12.
[14]张正杰,韩金秀,刘学周,等.内生菌株b16发酵条件优化及其对人参锈腐病的防效[J].微生物学通报, 2017, 44(11): 2698-2707.
[15]戴蓬博,蓝星杰,宗兆锋,等.极长链霉菌sl01菌株抑菌活性物质的发酵条件优化和稳定性研究[J]. 农药学学报, 2016, 18(6): 729-737.
[16]卢冰霞,何颖,赵武,等.应用正交试验优化猪细小病毒N株在IBRS-2细胞转瓶培养[J].西南农业学报, 2017, 30(2):839-842.
[17]邓志平,陈浩,陈三凤.利用响应面法优化固氮类芽孢杆菌paenibacillus sp.1-49的发酵培养基[J]. 微生物学报, 2016, 56(9): 1415-1425.
[18]Liu R S, Tang Y J. Tuber melanosporum fermentation medium optimization by Plackett-Burman design coupled with Draper-Lin small composite design and desirability function[J]. Bioresource Technology, 2010, 101(9): 3139-3146.
[19]洪亮,杨丽芬,杨建,等.小枣黑疔病拮抗放线菌的筛选与发酵优化[J].西南农业学报,2018,31(8):1634-1637.
[20]Sunitha K, Kim Y O, Lee J K, et al. Statistical optimization of seed and induction conditions to enhance phytase production by recombinant escherichia coli[J]. Biochemical Engineering Journal, 2000, 5(1): 51-56.
[21]黄艳,赵春田,裘娟萍.一株加纳链霉菌种子培养工艺及移种标准的研究[J]. 中国畜牧杂志, 2014, 50(7): 79-84.
[22]赵鑫,肖玉平,廖延智,等. L-精氨酸高产菌株的亚硝基胍诱变选育和种子培养基的优化研究[J].中国酿造, 2014, 33(9): 81-85.