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中国生物工程杂志

China Biotechnology
China Biotechnology
China Biotechnology  2017, Vol. 37 Issue (2): 54-62    DOI: 10.13523/j.cb.20170209
    
Production and Characteristics of Pullulan Produced by Aureobasidium pullulans A5
CHEN Guo-qiang1,2, WU Wen-tao2, WANG Ji-ming2, LIAO Wei-hong3, ZHANG Hai-bo2, XIAN Mo2, LEI Ting-zhou4, WEI Yu-xi1
1. College of Life Science, Qingdao University, Qingdao 266071, China;
2. CAS Key Laboratory of Biobased Materials, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao 266101, China;
3. Lukang Biological Manufacture Company Limited of Shandong, Zoucheng 273500, China;
4. Henan Academy of Sciences Institute of Energy Co., LTD, Zhengzhou 450000, China
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Abstract  

To screen strains which could selectively producing elevated molecular weight and pigment-free pullulan for the production of pullulan industry, a new strain was separated and then the culture condition was optimized. The improved YPD solid culture medium (addition of 1mg/L chloramphenicol and 0.5 g/L Trypen Blue) was used as selection medium. A new culture, strain A5, was screened and identified based on the morphology, colony and ITS interval sequence analysis. Single factor test was performed to optimize the fermentation conditions. The separation and purified production was identified and determined with Thin-layer chromatography (TLC), Fourier-transform infrared (FT-IR) and Gel permeation chromatography (GPC). The new strain was identified to be Aureobasidium pullulans, and named as A. pullulans A5, which could selectively produce pullulan. The fermentation conditions were optimized to be 8% (w/v) maltose, 1% (w/v) yeast extract, 2% (w/v) peptone, 0.5% (w/v) K2HPO4, 0.06% (w/v) (NH4)2SO4, 0.03% (w/v) CaCl2, initial pH6, 7% (v/v) inoculation amount. TLC and FT-IR spectroscopy of this production were consistent with those of the standard, and the molecular weight was 63.84 kDa. A yeast-like fungal strain A5 was successfully obtained and identified with its high molecular weight and pigment-free production. At the optimized conditions, the pullulan yield was 22.9 g/L. So a candidate strain for the production of pullulan industry was provided.

Key words： Pullulan      Structural identification      Molecular weight      Aureobasidium pullulans      Fermentation condition     
Received: 09 September 2016      Published: 25 February 2017
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CHEN Guo-qiang, WU Wen-tao, WANG Ji-ming, LIAO Wei-hong, ZHANG Hai-bo, XIAN Mo, LEI Ting-zhou, WEI Yu-xi. Production and Characteristics of Pullulan Produced by Aureobasidium pullulans A5. China Biotechnology, 2017, 37(2): 54-62.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.20170209     OR     https://manu60.magtech.com.cn/biotech/Y2017/V37/I2/54

[1] Cheng K, Demirci A, Catchmark J M. Pullulan:biosynthesis, production, and applications. Applied Microbiology Biotechnology, 2011, 92(1):29-44.
[2] 刘谋泉.普鲁兰多糖发酵条件研究.食品工业科技,2008,29(1):110-112. Liu M Q. Study on fermentation conditions of pullulan. Science and Technology of Food Intustry, 2008, 29(1):110-112.
[3] 邵伟,刘世玲,唐明,等.茁霉多糖发酵及提取工艺条件研究. 生物技术,2004, 14(5):69-70. Shao W, Liu S L, Tang M, et al. Studies on fermentation and purification of pullulan. Biotechnology, 2004, 14(5):69-70.
[4] Lee J, Kim J, Zhu I, et al. Optimization of conditions for the production of pullulan and high molecular weight pullulan by Aureobasidium pullulans. Biotechnology Letters, 2001, 23:817-820.
[5] Ono K, Yasuda N. Effect of pH on pullulan elaboration by Aureobasidium pullulans S-l. Agricultural Biological Chemistry, 1977, 41(11):2113-2118.
[6] Lacroixt C, Leduy A, Noel G, et al. Effect of pH on the batch fermentation of Pullulan from sucrose medium. Biotechnology Bioenggineering, 1985,27(2):202-207.
[7] Bulmer M A, Catley B J, Kelly P J. The effect of ammonium ions and pH on the elaboration of the fungal extracellular polysaccharide, pullulan, by Aureobasidium pullulans. Applied Microbiology Biotechnology, 1987, 25:362-365.
[8] Wu S, Jin Z, Tong Q, et al. Sweet potato:A novel substrate for pullulan production by Aureobasidium pullulans. Carbohydrate Polymers, 2009, 76(4):645-649.
[9] 李世杰,彭华松,方尚玲.玉米淀粉为原料短梗霉多糖发酵条件研究.粮食与饲料工业, 2000,12:41-42. Li S J, Peng H S, Fang S L. Study on pullulan fermentation with corn starch as culture material. Food and Feed Industry, 2000, 12:41-42.
[10] Vijayendra S V, Bansal D, Prasad M S, et al. Jaggery:a novel substrate for pullulan production by Aureobasidium pullulans CFR-77. Process Biochemistry, 2001, 37:359-364.
[11] 韩丛琴,王兴华.不同微量元素对出芽短梗霉发酵的影响.山东农业科学,2012, 44(3):112-114. Han C Q, Wang X H. Effects of different microelements on Aureobasidium pullulans fermentation. Shandong Agricultural Sciences, 2012, 44(3):112-114.
[12] 鞠宝,陈永珉,林剑,等.二价阳离子对短梗霉多糖发酵的影响.生物技术,2000, 10(4):31-33. Ju B, Chen Y M, Lin J, et al. Effects of two valanceions on pullulan fermentation. Biotechnology, 2000, 10(4):31-33.
[13] 牛登飞,童群义.金属离子对短梗霉多糖发酵的影晌.食品工业科技,2009, 30(9):154-157. Niu D F, Tong Q Y. Study on effects of metal ions on pullulan production by Aureobasidium pullulans. Science and Technology of Food Industry, 2009, 30(9):154-157.
[14] Kossaczká Z, Machová E, Vojtková-Lepšíková A. D-Xylose metabolism in Aureobasidium pullulans:effects of aeration and vitamins. Applied Microbiology Biotechnology, 1991, 36:375-378.
[15] 梁雪.产胞外多糖酵母菌株的筛选鉴定及多糖结构初步分析。哈尔滨:东北林业大学硕士学位论文,2010. Liang X. Screening, identification of exopolysaccharide-producing yeasts and studies on exopolysaccharide structure elementarily. Haerbin:Master Degree Thesis of Northeast Forestry University, 2010.
[16] 魏景超.真菌鉴定手册.上海:上海科学技术出版社,1979.103-117. Wei J C. The Manual of Determinative Mycology. Shanghai:Shanghai Scientific & Technical Publishers, 1979. 103-117.
[17] Dong X, Yang J, Zhu N, et al. Sugarcane bagasse degradation and characterization of three white-rot fungi. Bioresource Technology, 2013, 131:443-451.
[18] Lazaridou A, Biliaderis C G, Roukas T, et al. Production and characterization of pullulan from beet molasses using a nonpigmented strain of Aureobasidium pullulans in batch culture. Applied Biochemistry Biotechnology, 2002, 97(1):1-22.
[19] Duan X, Chi Z, Wang L, et al. Influence of different sugars on pullulan production and activities of α-phosphoglucose mutase, UDPG-pyrophosphorylase and glucosyltransferase involved in pullulan synthesis in Aureobasidium pullulans Y68. Carbohydrate Polymers, 2008, 73(4):587-593.
[20] 曹海石,孙宏,梁放,等.普鲁兰多糖的分离纯化及结构鉴定.高等学校化学学报,1999, 20(11):1729-1732. Cao H S, Sun H, Liang F, et al. Prification and determination of the structure of pullulan polysaccharide. Chemical Journal of Chinese Universities, 1999, 20(11):1729-1732.
[21] 吴少微.鸡骨草多糖的分离纯化、结构分析和抗氧化活性. 广州:华南理工大学硕士学位论文,2013. Wu S W. Preparation, preliminary charecterization and antioxidant activities of polysaccharides from Abrus cantoniensis Hance. Guangzhou:Master Degree Thesis of South China University of Technology, 2013.
[22] 陈萌.四株不同来源海洋微生物胞外多糖的结构及抗氧化活性研究. 青岛:中国海洋大学博士学位论文,2012. Chen M, Structural charecterization and antioxidant properties of exopolysaccharides produced by 4 marine microbe with different sources. Qingdao:Doctoral Dissertation of Ocean University of China, 2012.
[23] Gadd G M. Melanin production and differentiation in batch cultures of the polymorphic fungus Aureobasidium pullulan. FEMS Microbiology Letters, 1980, 9:237-240.
[24] West T P, Reed-Hamer B. Polysaccharide production by a reduced pigmentation mutant of the fungus Aureobasidium pullulans. FEMS Microbiology Letters, 1993, 113(3):345-349.
[25] 张忠智,将令西,王利涛,等.生物多糖代谢产物的调控过程优化研究. 化学与生物工程,2005, 11:36-38. Zhang Z Z, Jiang L X, Wang L T, et al. Optimization research on the regulation of biopolysaccharides metabolic product. Chemistry & Bioengineering, 2005, 11:36-38.
[26] Lazaridou A, Biliaderis C G, Roukas T, et al. Production and characterization of pullulan from beet molasses using a nonpigmented strain of Aureobasidium pullulans in batch culture. Biochemistry and Biotechnology, 2001, 97:1-22.
[27] West T P, Reed-Hamer B. lnfluence of vitamins and mineral salts upon pullulan synthesis by Aureobasidium pullulans. Microbios, 1992, 71:115-123.
[28] 周瑞芳,荚恒龙,彭风鼐.稻米中普鲁兰酶的纯化与性质的研究.中国粮油学报,1994, 9(1):11-16. Zhou R F, Jia H L, Peng F N. Study on the purification and characterizations of pullulanase in rice. Journal of the Chinese Cereals and Oils Association, 1994, 9(1):11-16.
[29] 谢晶曦.红外光谱在有机化学和药物化学中的应用.北京:北京科学出版社,1987. 77. Xie J X. Applications of Infrared Spectroscopy in Organic Chemistry and Pharmaceutical Chemistry. Beijing:Beijing Science Press, 1987. 77.
[30] Kim J, Kim M, Lee J, et al. Production of high molecular weight pullulan by Aureobasidium pullulans using glucosamine. Biotechnology Letters, 2000, 22:987-990.
[31] Israilides C, Scanlon B, Smith A, et al. Characterization of pullulans produced from agro-industrial wastes. Carbohydrate Polymers, 1994, 25:203-209.
[32] Kachhawa D K, Bhattacharjee P, Singhal R S. Studies on downstream processing of pullulan. Carbohydrate Polymers, 2003, 52:25-28.
[33] Li B, Zhang N, Peng Q, et al. Production of pigment-free pullulan by swollen cell in Aureobasidium pullulans NG which cell differentiation was affected by pH and nutrition. Applied Microbiology Biotechnology, 2009, 84(2):293-300.
[34] Zheng W, Campbell B S, McDougall B M, et al. Effects of melanin on the accumulation of exopolysaccharides by Aureobasidium pullulans grown on nitrate. Bioresource Technology, 2008, 99(16):7480-7486.
[35] Wei G, Yu X, Wang Y, et al. Media optimization for elevated molecular weight and mass production of pigment-free pullulan. Carbohydrate Polymers, 2012, 89(3):928-934.
[36] Roukas T, Youssef F, Biliaderis C G. Pullulan production by a non-pigmented strain of Aureobasidium pullulans using batch and fed-batch culture. Process Biochemistry, 1999, 34:355-366.
[37] Youssef F, Biliaderis C G, Roukas T. Enhancement of pullulan production by Aureobasidium pullulans in batch culture using olive oil and sucrose as carbon sources. Applied Biochemistry Biotechnology, 1998, 74(74):13-30.
[38] 胡洪森, 叶斌.溴乙锭对出芽短梗霉的诱变作用.武汉生物工程学院学报,2012, 8(2):79-81. Hu H S, Ye B. Mutagenic effect of ethidium bromide on Aureobasidium pullulans. Journal of Wuhan Bioengineering Institude, 2012, 8(2):79-81.
[39] Singh R S, Saini G K, Kennedy J F. Downstream processing and characterization of pullulan from a novel colour variant strain of Aureobasidium pullulans FB-1. Carbohydrate Polymers, 2009, 78(1):89-94.
[40] 曹新志.普鲁兰的生产和应用研究进展.四川理工学院学报,2006, 19(4):60-62. Cao X Z. Development of production and applications of pullulan. Journal of Sichuan University of Science & Engineering, 2006, 19(4):60-62.
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