优化根瘤菌(Rhizobium NG10)产多糖的培养条件,提高胞外多糖(extracelluar polysaccharides of Rhizobium,REPS)产量,并探究其抗肿瘤活性。通过单因素实验、Plackett-Burman实验和Box-Behnken实验等对根瘤菌产胞外多糖发酵培养基配方进行优化,并采用MTT法检测多糖对人肝癌细胞的抑制作用。最终优化得到的最优培养基配方为:麦芽糖22.5 g/L,大豆蛋白胨9.5 g/L,MgSO4·7H2O 0.2 g/L,KH2PO4 0.41 g/L,NaCl 0.1 g/L,此时胞外多糖产量为5.05 g/L,相比原始提高了45.95 %;REPS对肝癌细胞Hep G2的抑制呈一定的量效关系,培养时间为48 h时IC50为393.3 μg/mL,肝癌细胞抑制率达到56.74 %。
In this study, the optimal the culture conditions for production of extracellular polysaccharides by Rhizobium sp. NG10 were determined. Besides, the inhibitory effect of the polysaccharides on human hepatocellular carcinoma cells was studied by MTT assay. The optimal medium composition for extracellular polysaccharides production were 22.5 g/L maltose, 9.5 g/L soybean peptone, 0.2 g/L MgSO4·7H2O, 0.41 g/L KH2PO4 and 0.1 g/L NaCl. Under these conditions, the yield of the polysaccharides was increased 45.95% with titer of 5.05 g/L. Moreover, the inhibitory effect of the polysaccharides on human hepatocellular carcinoma cells exhibited a dose-effect relationship. When the human hepatocellular carcinoma cells were cultured with the polysaccharides for 48 h, the IC50 was 393.3 μg/mL, and the inhibition rate was 56.74%.
[1] GHOSH A C, GHOSH S,BASU P S, et al. Production of extracellular polysaccharide by a rhizobium species from root nodules of the leguminous tree Dalbergia lanceolaria[J]. Engineering in Life Sciences, 2010, 5(4):378-382.
[2] 冯永君,宋未. 根瘤菌胞外多糖的结构与信号功能[J]. 生命的化学, 2000,20(1):33-35.
[3] 钱世钧,雨村明伦,原田笃也. 根瘤菌TISTR386胞外酸性多糖的结构研究[J]. 微生物学报, 1984,24(4):45-54.
[4] CHENG H B, HAN Y, REN S, et al. Studies on isolation and anti-S180 activity of exopolysaccharide from Rhizobium sp.N613[J]. Chinese Pharmaceutical Journal, 2008, 43(4):268-272.
[5] WANG X, SHAO C, LIU L, et al. Optimization, partial characterization and antioxidant activity of an exopolysaccharide from, Lactobacillus plantarum KX041[J]. International Journal of Biological Macromolecules, 2017,103:1 173-1 184.
[6] PRIYANKA P, ARUN A B, ASHWINI P, et al. Versatile properties of an exopolysaccharide R-PS18 produced by Rhizobium sp. PRIM-18[J]. Carbohyd Polym, 2015,126:215-221.
[7] 陈艳丽,任盛,魏国琴,等. Rhizobium sp.N613胞外多糖的抗氧化活性[J]. 微生物学通报, 2010, 37(2):234-238.
[8] PAN D, LIU J, ZENG X, et al. Immunomodulatory activity of selenium exopolysaccharide produced by Lactococcus lactis subsp. Lactis[J]. Food and Agricultural Immunology, 2015, 26(2):248-259.
[9] 龙寒,陈盛峰,陈佳,等. 一株产胞外多糖海洋弧菌的分离鉴定及其多糖抗肿瘤活性初步研究[J]. 生物技术通报, 2016,32(12):166-171.
[10] 黄晓波,张建国,韩勇,等. 一种根瘤菌胞外多糖对小鼠免疫功能的影响[J]. 免疫学杂志, 2006, 22(S1):149-150.
[11] 韩勇,黄晓波,董岳峰,等. 根瘤菌N613胞外多糖发酵条件及抗肿瘤作用研究[J]. 微生物学通报, 2007, 34(5):909-913.
[12] GHOSH A C, GHOSH S, BASU, P S. Production of extracellular polysaccharide by a Rhizobium species from root nodules of the leguminous tree Dalbergia lanceolaria[J]. Engineering in Life Sciences, 2010, 5(4):378-382.
[13] 吴迪,袁峰,王国瑞,等. 基于通气量调控的灵芝菌丝体胞内多糖发酵工艺优化[J]. 食用菌学报,2019,26(1):35-40.
[14] 王萧玉竹,董晋军,许国超,等. 可得然胶生产菌种的筛选及发酵条件优化[J]. 食品与生物技术学报,2018,37(7):732-738.
[15] CHAMBON R, PRADEAU S, FORT S, et al. High yield production of Rhizobium NodB chitin deacetylase and its use for in vitro synthesis of lipo-chitinoligosaccharide precursors[J]. Carbohydrate Research, 2017, 442:25-30.
[16] 戎蓉,欧杰. 响应面法优化普鲁兰多糖发酵工艺条件[J]. 山东农业大学学报(自然科学版), 2016, 47(5):659-663.
[17] 褚以文. 微生物培养基优化方法及其OPTI优化软件[J]. 国外医药(抗生素分册), 1999,20(2):58-60.
[18] 赵刚,刘振华,辛明杨,等. Plackett-Burman设计及响应面优化蓝莓中总黄酮的超声提取工艺[J]. 食品工业, 2017,38(10):22-27.
[19] 王云龙,刘松,堵国成,等. 基于人工神经网络的L-天冬酰胺酶发酵培养基优化[J]. 食品与发酵工业, 2018, 44(8):27-33.
[20] CHIEF E I, DEMAIN A L, ESITORS J E D, et al. Manual of Industrial Microbiology and Biotechnology[M]. Washington DC: American Society for Microbiology, 1986.
[21] BORKOWSKI J J. Minimum, maximum, and average spherical prediction variances for central composite and Box-Behnken designs[J]. Communications in Statistics-Theory and Methods, 1995, 24(10):2 581-2 600.
[22] 于士军. 蝙蝠蛾拟青霉胞外多糖活性和结构的研究[D].合肥: 安徽农业大学, 2012.
