生产与科研应用

产胞外多糖的苏云金芽孢杆菌的筛选及发酵工艺优化

  • 杨静 ,
  • 高泽鑫 ,
  • 朱莉 ,
  • 詹晓北
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  • 1(糖化学与生物技术教育部重点实验室,江南大学 生物工程学院,江苏 无锡,214122)
    2(无锡格莱克斯生物科技有限公司,江苏 无锡,214125)
硕士研究生(詹晓北教授为通讯作者,E-mail:xbzhan@yahoo.com)

收稿日期: 2020-03-24

  修回日期: 2020-04-01

  网络出版日期: 2022-01-21

基金资助

十三五国家重点研发技术(2017YFD0400302)

Screening of an extracellular polysaccharides producing Bacillus thuringiensis strain and its fermentation optimization

  • YANG Jing ,
  • GAO Zexin ,
  • ZHU Li ,
  • ZHAN Xiaobei
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  • 1(Key Laboratory of Carbohydrate Chemistry and Biotechnology of Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China)
    2(Wuxi Galaxy Biotech Co.Ltd., Wuxi 214125, China)

Received date: 2020-03-24

  Revised date: 2020-04-01

  Online published: 2022-01-21

摘要

微生物胞外多糖(exopolysaccharidess,EPS)因其生产周期短等优点具有潜在的应用价值。该文从纳豆中筛选出1株高产EPS的菌株并鉴定该菌株为苏云金芽孢杆菌(Bacillus thuringiensi)。为提高其EPS产量,通过对发酵培养基的组分和发酵培养条件进行单因素试验,然后采用Plackett-Burman(PB)设计对9个因素的重要性进行评判,从中选出3个重要影响因素进行Box-Benhnken实验设计优化得到最终的最佳培养方案:葡萄糖22.6 g/L,黄豆饼粉9.3 g/L,玉米浆2.0 g/L,K2HPO4 0.5 g/L,NaH2PO4 1.5 g/L,MgSO4 1.5 g/L,MnCl2 0.05 g/L,初始pH 7.5,装液量70 mL,温度为34 ℃,接种量为6%。与初始发酵工艺相比,EPS产量提高了133.3%。经7 L发酵罐放大验证后,EPS产量增加至6.56 g/L,与摇瓶水平相比,EPS产量提高了123.1%,并且已初步探索该EPS具有纳豆激酶活性。有望成为合成药物的潜在来源。

本文引用格式

杨静 , 高泽鑫 , 朱莉 , 詹晓北 . 产胞外多糖的苏云金芽孢杆菌的筛选及发酵工艺优化[J]. 食品与发酵工业, 2021 , 47(24) : 124 -131 . DOI: 10.13995/j.cnki.11-1802/ts.027440

Abstract

Microbial exopolysaccharides (EPS) has potential application value due to its short production cycle and other advantages. In this paper, a strain with high yield of EPS was screened from natto and identified as Bacillus thuringiensi. To improve its EPS production, the fermentation medium and fermentation conditions of strain were optimized by single-factor experiments. The Plackett-Burman (PB) design was used to evaluate the importance of nine factors, and three important factors were selected. Box-Benhnken experimental design was used to get the final optimal culture: 22.6 g/L glucose, 9.3 g/L soybean meal, 2.0 g/L corn syrup, 0.5 g/L K2HPO4, 1.5 g/L NaH2PO4, 1.5 g/L MgSO4, 0.05 g/L MnCl2, pH 7.5, loading volume 70 mL, 34 ℃, inoculum size of 6%. Compared with the initial fermentation process, the EPS production increased by 133.33%. Using 7 L fermenter to amplify and verify, the yield of EPS increased to 6.56 g/L. Compared with the shake flask level, the EPS production increased by 123.1% and the EPS had nattokinase activity. It is expected to become a potential source of synthetic drugs.

参考文献

[1] FREITAS F, TORRES C A V, REIS M A M.Engineering aspects of microbial exopolysaccharide production[J].Bioresource Technology, 2017, 245:1 674-1 683.
[2] WANG J, SALEM D R, SANI R K.Extremophilic exopolysaccharides:A review and new perspectives on engineering strategies and applications[J].Carbohydrate Polymers, 2019, 205:8-26.
[3] LIU J, WANG X C, PU H M, et al.Recent advances in endophytic exopolysaccharides:Production,structural characterization, physiological role and biological activity[J].Carbohydrate Polymers, 2017, 157(10):1 113-1 124.
[4] YILDIZ H, KARATAS N.Microbial exopolysaccharides:Resources and bioactive properties[J].Process Biochemistry, 2018, 72:41-46.
[5] SHUKLA A, MEHTA K, PARMAR J, et al.Depicting the exemplary knowledge of microbial exopolysaccharides in a nutshell[J].European Polymer Journal, 2019, 119:298-310.
[6] MALICK A, KHODAEI N, BENKERROUM N, et al.Production of exopolysaccharides by selected Bacillus strains:Optimization of media composition to maximize the yield and structural characterization[J].International Journal of Biological Macromolecules, 2017, 102:539-549.
[7] AMIRI S, REZAEI MOKARRAM R, SOWTI KHIABANI M, et al.Exopolysaccharides production by Lactobacillus acidophilus LA5 and Bifidobacterium animalis subsp.lactis BB12:Optimization of fermentation variables and characterization of structure and bioactivities[J].International Journal of Biological Macromolecules, 2019, 123(15):752-765.
[8] 李琪雯, 周嫄, 柯成竹, 等.微生物生产威兰胶的研究进展[J].食品工业科技, 2019, 40(23):337-342;348.
LI Q W, ZHOU Y, KE C Z, et al.Research progress in microbial production of welan gum[J].Science and Technology of Food Industry, 2019, 40(23):337-342;348.
[9] ZHOU Y, CUI Y H, QU X J.Exopolysaccharides of lactic acid bacteria:Structure, bioactivity and associations:A review[J].Carbohydrate Polymers, 2019, 207:317-332.
[10] ZHONG C Y, CAO G, RONG K, et al.Characterization of a microbial polysaccharide-based bioflocculant and its anti-inflammatory and pro-coagulant activity[J].Colloids and Surfaces B:Biointerfaces, 2018, 161(1):636-644.
[11] OERLEMANS M M P, AKKERMAN R, FERRARI M, et al.Benefits of bacteria-derived exopolysaccharides on gastrointestinal microbiota, immunity and health[J].Journal of Functional Foods, 2021, 76:104289.
[12] RAMAMOORTHY S, GNANAKAN A, S. LAKSHMANA S, et al.Structural characterization and anticancer activity of extracellular polysaccharides from ascidian symbiotic bacterium Bacillus thuringiensis[J].Carbohydrate Polymers, 2018, 190(15):113-120.
[13] 张红艳, 李忠玲, 张强, 等.地衣芽孢杆菌MYS68的鉴定及发酵培养基优化[J].粮食与饲料工业, 2018(2):50-53.
ZHANG H Y, LI Z L, ZHANG Q, et al.Identification and optimization of fermentation medium for Bacillus licheniformis MYS 68[J].Cereal & Feed Industry, 2018(2):50-53.
[14] 高泽鑫, 何腊平, 刘亚兵, 等.纳豆激酶的研究进展与展望[J].中国酿造, 2017, 36(8):11-15.
GAO Z X, HE L P, LIU Y B, et al.Research progress and prospect of nattokinase[J].China Brewing, 2017, 36(8):11-15.
[15] 刘卫宝, 余讯, 徐静静, 等.黄芪多糖的分离、结构表征及益生活性研究[J].食品与发酵工业, 2020, 46(7):50-56.
LIU W B, YU X, XU J J, et al.Isolation, structure characterization and prebiotic activity of polysaccharides from Astragalus membranaceus[J].Food and Fermentation Industries, 2020, 46(7):50-56.
[16] 陈博文, 李贞蓉, 常明昌, 等.香菇产α-半乳糖苷酶的液体发酵工艺优化[J].食用菌学报, 2018, 25(2):79-89.
CHEN B W, LI Z R, CHANG M C, et al.Optimization of fermention conditions for the production of α-galactosidase from Lentinula edodes[J].Acta Edulis Fungi, 2018, 25(2):79-89.
[17] 高泽鑫. 高产纳豆激酶菌株的筛选及其酶学稳定性的研究[D].贵阳:贵州大学, 2018.
GAO Z X.Screening of high-yield nattokinase strains and study of its enzymatic stability[D].Guiyang:Guizhou University, 2018.
[18] 李梅云, 高家合, 王革, 等.苏云金杆菌伴孢晶体形态特征观察[J].烟草科技, 2004, 208(11):43-45.
LI M Y, GAO J H, WANG G, et al.Morphologic characteristics of parasporal crystals of Bacillus thuringiensis[J].Tobacco Science & Techonlogy/Disease & Pest Control, 2004, 208(11):43-45.
[19] 杨树丽. 一株产胞外多糖芽孢杆菌的研究[D].上海:上海应用技术大学, 2016.
YANG S L.Study on Bacillus strains producing extracellular polysaccharide[D].Shanghai:Shanghai Institute of Technology, 2016.
[20] R.E.布坎南.伯杰氏细菌鉴定手册[M].第八版.北京:科学出版社, 1984:1 668.
R.E.BUCHANAN.BergeÝs Manual of Determinative Bacteriology[M].8th ed.Beijing:Science Press, 1984:1 668.
[21] ATTESON K.The performance of neighbor-joining methods of phylogenetic reconstruction[J].Algorithmica, 1999, 25(2-3):251-278.
[22] 张路路, 朱朝华, 郭刚.苏云金芽孢杆菌A322菌株发酵培养基和发酵条件的优化[J].热带生物学报, 2014, 5(3):253-259.
ZHANG L L, ZHU C H, GUO G.Optimization of Bacillus thuringiensis A322 strain fermentation medium and cultural conditions[J].Journal of Tropical Biology, 2014, 5(3):253-259.
[23] 胡红伟, 段明房, 闫凌鹏, 等.一株枯草芽孢杆菌的鉴定及液体发酵工艺优化[J].中国饲料, 2017(5):13-19.
HU H W, DUAN M F, YAN L P, et al.Identification of a Bacillus subtilis strain and optimization of its liquid fermentation process[J].China Feed, 2017(5):13-19.
[24] 庞远祥, 谢远红, 金君华, 等.低嘌呤、高纳豆激酶活性枯草芽孢杆菌SH21筛选及发酵条件优化[J].食品与发酵工业, 2021,47(11):194-199.
PANG Y X, XIE Y H, JIN J H, et al.Isolation and optimization of Bacillus subtilis SH21 for low purine and high nattokinase activity[J].Food and Fermentation Industries, 2021,47(11):194-199.
[25] CAGRI-MEHMETOGLU A, KUSAKLI S, VENTER V.Production of polysaccharide and surfactin by Bacillus subtilis ATCC 6633 using rehydrated whey powder as the fermentation medium[J].Journal of Dairy Science, 2012, 95(7):3 643-3 649.
[26] ASGHER M, UROOJ Y, QAMAR S A, et al.Improved exopolysaccharide production from Bacillus licheniformis MS3:Optimization and structural/functional characterization[J].International Journal of Biological Macromolecules, 2020, 151:984-992.
[27] SUSAN V D J, KEE N L A, FROST C L, et al.Extracellular polysaccharide production in Bacillus Licheniformis SVD 1 and its immunomodulatory effect[J].Bioresources, 2012, 7(4):4 976-4 993.
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