新型微生物多糖威兰胶的发酵研究
摘要
威兰胶(Welan Gum、又名威伦胶或韦兰胶)是由细菌发酵生产的胞外杂多糖,过去的名称为S-130。威兰胶是美国的Kelco公司上世纪80年代继黄原胶和结冷胶之后开发的最具市场前景的微生物代谢多糖之一。因其特定的流变性和独特的分子结构而在石油开采、建筑、食品等领域有广泛的应用开发价值。
研究了威兰胶的发酵特性,优化了发酵培养基的组成和发酵培养条件,探讨了威兰胶的提取、分离和纯化工艺,对威兰胶样品的分子结构进行了初步的探索研究,最后对制得的威兰胶样品进行了流变学性质的研究。主要的研究结果如下:
本研究以威兰胶产量和黏度为指标进行了发酵工艺的优化,并研究了其代谢规律。确定发酵培养条件为:40g/L蔗糖,4g/L复合氮源(20%硝酸钠和80%的牛肉膏),2g/L KH_2PO_4,0.1g/L MgSO_4,0.5mL/L的0.1%FeSO_4。初始pH为7.2～7.4,5%的接种量、装液量为50mL/250mL三角瓶、转速为200r/min、30℃恒温培养58h,威兰胶产量从9.326g/L提高到21.767g/L。菌体在培养到对数生长期中后期接种,即16h～24h,而大约44小时后进入衰亡期,生物量缓慢下降,威兰胶产量迅速上升,因此发酵时最好在16h～24h接种,可以避免或缩短停滞期,缩短发酵时间。通过对威兰胶粗品的预处理和进一步的分离纯化工艺,得到了纯化样品,用传统化学方法、薄层层析、离子色谱、红外光谱、气质联用等对其化学结构进行了研究,分析得威兰胶结构中含有鼠李糖、葡萄糖、甘露糖、半乳糖和葡萄糖醛酸,百分比分别约为:36.06%,32.58%,8.98%,5.28%,14.33%。总糖含量84.95%,乙酰基含量为5.8375%;糖苷键类型主要为B构型。
对威兰胶流变学性质进行了初步的研究,确定了成兰胶水溶液为高假塑性流体,其黏度在不同的温度(30℃～121℃),浓度,pH范围(2～12),盐离子(钠、钾、钙、铝)等外在环境的影响下极其稳定,与黄原胶比较具有更好的效果。
Welan gum is a heteropolysaccharide produced by Alcaligenes ATCC 31555(previously named S-130). The unique feature of welan, rheology property and its special molecular structure has attracted the extensive attention of oil well drilling fluids, Building Industry and food industry. In this thesis, we studied fermentation characters of welan; the ideal culture medium and fermentation conditions. The progresses of isolation, purification and separation of welan gum from fermentation broth were studied, and the structure of the purified welan gum which was separated and purified from fermentation broth was analyzed. The rheological property of welan gum solution was studied. The main Results were as following:
According to the yield and viscosity of welan gum, the optimal parameters of fermentation were studied. Results showed that the optimal fermentation medium contained: 40g/L sucose,4g/L complex nitrogen (20% sodium nitrate and 80% beef extract), 2g/L KH_2PO_4, 0.1g/L MgSO_4 and 0.5mL/L 0.1% FeSO_4. The optimized condition were: initial pH 7.2～7 .4, inoculation volume 5%, and aeration ratio 1:5(medium volume: flask volume). 30℃for 58h in 250mL shake flasks at 200r/min, the yield of welan gum increased from 9.326g/L to 21.767 g/L. The anaphase of logarithm growth of the strains is 16h～24h, it enter into contabescence about 44h, from that, mycelium biomass declining slowly. Therefore, it should be better to inoculate at 16h～424h, the stagnation of mycelium growth can be avided and fermentation period was shorten.
According to pretreatment of the crude Welan gum and farther separation and purification, the analytic sample was obtained. The components of analytic sample were identified by traditional chemical method、TLC、Ion chromatography、IR、and GC-MS, results showed that it contained rhamnose, glucose, mannose, galactose,and glucuronic acid, the respective percentages are 36.06%, 32.58%, 8.98%, 5.28%, 14.33%. The content of saccharide and acetyl is 84.95%、5.8375%, respectively. The polysaccharide was mainly consisting of (3 glucosidic linkages.
The rheological property of welan gum solution was studied. It showed that the solution of welan gum is a typical pseudoplastic liquid. Under the external influence of the environment, which has included different temperatures (30℃～121℃), concentration, pH (2～12) and salt (sodium, potassium, calcium, aluminum), viscosity is extremely stable. Welan Gum showed widely applications future compared with xanthan gum.
研究了威兰胶的发酵特性,优化了发酵培养基的组成和发酵培养条件,探讨了威兰胶的提取、分离和纯化工艺,对威兰胶样品的分子结构进行了初步的探索研究,最后对制得的威兰胶样品进行了流变学性质的研究。主要的研究结果如下:
本研究以威兰胶产量和黏度为指标进行了发酵工艺的优化,并研究了其代谢规律。确定发酵培养条件为:40g/L蔗糖,4g/L复合氮源(20%硝酸钠和80%的牛肉膏),2g/L KH_2PO_4,0.1g/L MgSO_4,0.5mL/L的0.1%FeSO_4。初始pH为7.2～7.4,5%的接种量、装液量为50mL/250mL三角瓶、转速为200r/min、30℃恒温培养58h,威兰胶产量从9.326g/L提高到21.767g/L。菌体在培养到对数生长期中后期接种,即16h～24h,而大约44小时后进入衰亡期,生物量缓慢下降,威兰胶产量迅速上升,因此发酵时最好在16h～24h接种,可以避免或缩短停滞期,缩短发酵时间。通过对威兰胶粗品的预处理和进一步的分离纯化工艺,得到了纯化样品,用传统化学方法、薄层层析、离子色谱、红外光谱、气质联用等对其化学结构进行了研究,分析得威兰胶结构中含有鼠李糖、葡萄糖、甘露糖、半乳糖和葡萄糖醛酸,百分比分别约为:36.06%,32.58%,8.98%,5.28%,14.33%。总糖含量84.95%,乙酰基含量为5.8375%;糖苷键类型主要为B构型。
对威兰胶流变学性质进行了初步的研究,确定了成兰胶水溶液为高假塑性流体,其黏度在不同的温度(30℃～121℃),浓度,pH范围(2～12),盐离子(钠、钾、钙、铝)等外在环境的影响下极其稳定,与黄原胶比较具有更好的效果。
Welan gum is a heteropolysaccharide produced by Alcaligenes ATCC 31555(previously named S-130). The unique feature of welan, rheology property and its special molecular structure has attracted the extensive attention of oil well drilling fluids, Building Industry and food industry. In this thesis, we studied fermentation characters of welan; the ideal culture medium and fermentation conditions. The progresses of isolation, purification and separation of welan gum from fermentation broth were studied, and the structure of the purified welan gum which was separated and purified from fermentation broth was analyzed. The rheological property of welan gum solution was studied. The main Results were as following:
According to the yield and viscosity of welan gum, the optimal parameters of fermentation were studied. Results showed that the optimal fermentation medium contained: 40g/L sucose,4g/L complex nitrogen (20% sodium nitrate and 80% beef extract), 2g/L KH_2PO_4, 0.1g/L MgSO_4 and 0.5mL/L 0.1% FeSO_4. The optimized condition were: initial pH 7.2～7 .4, inoculation volume 5%, and aeration ratio 1:5(medium volume: flask volume). 30℃for 58h in 250mL shake flasks at 200r/min, the yield of welan gum increased from 9.326g/L to 21.767 g/L. The anaphase of logarithm growth of the strains is 16h～24h, it enter into contabescence about 44h, from that, mycelium biomass declining slowly. Therefore, it should be better to inoculate at 16h～424h, the stagnation of mycelium growth can be avided and fermentation period was shorten.
According to pretreatment of the crude Welan gum and farther separation and purification, the analytic sample was obtained. The components of analytic sample were identified by traditional chemical method、TLC、Ion chromatography、IR、and GC-MS, results showed that it contained rhamnose, glucose, mannose, galactose,and glucuronic acid, the respective percentages are 36.06%, 32.58%, 8.98%, 5.28%, 14.33%. The content of saccharide and acetyl is 84.95%、5.8375%, respectively. The polysaccharide was mainly consisting of (3 glucosidic linkages.
The rheological property of welan gum solution was studied. It showed that the solution of welan gum is a typical pseudoplastic liquid. Under the external influence of the environment, which has included different temperatures (30℃～121℃), concentration, pH (2～12) and salt (sodium, potassium, calcium, aluminum), viscosity is extremely stable. Welan Gum showed widely applications future compared with xanthan gum.
引文
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[31]杨仁智,张劲松,唐庆九.高效阴离子交换色谱法测定毛头鬼伞多糖中的单糖组成[J].中国食用菌,2005,24(5):42-44
[32]张培敏,朱岩,凌艳艳.离子色谱脉冲安培检测法测定水体颗粒物与河口沉积物中的糖[J].浙江大学学报,2004,31(4):431-434
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[3]LanW.Sutherland.Novel and established applications of microbial polysaccharides[J].TIBTECH.1998,16(1):41-46
[4]胡国华.功能性食品胶[M].北京:化学工业出版社,2004:216-217
[5]魏培莲.微生物胞外多糖的研究进展[J].浙江科技学院学报,2002,14(11):8-12
[6]黄原胶生产技术[J],中国科技产业,2003,3:81-82
[7]刘清泉.黄原胶产业的线装及发展趋势[J],中国食品添加剂,2002,6:5-7
[8]Kang K S,Veeder G T.Heteropolysaccharide S-130[P]US:4342866,1981
[9]Jansson P E,Widmalm G.Welan gum(S-130) contains repeating units with randomly distributed L-mannosyl and L-rhamnosyl terminal groups,as determined by FABMS[J].Carbohydr.Res,1994,256(2):327-330
[10]Stankowski J D,Zeller S G..Location of the O-acetyl group in welan by the reductive-cleavage method[J].Carbohydr.Res.,1992,224,337-341
[11]Tako,M,Kiriaki,M.Rheological properities of welan gum in aqueous media[J].Agric.Biol.Chem 1990,54(12):3079-3084
[12]陈代杰、金飞燕主译.生物大分子(第5卷)化学工业出版社,2004:250-254
[13]张晨,刘志伟.糖的生物合成及微生物多糖发酵,嘉应大学学报,1999,3:88-91
[14]Sutherland IW.Microbial exopolysaccharide synthesis.In:Sandford PA,Laskin A,editors Extracellular microbial polysaccharides[M].Washington,DC:American Chemical Society.p40-57,1977
[15]李莎.威兰胶发酵工艺优化及结构性能的研究[D].南京:南京工业大学,2005
[16]Lea Lopes,Michel Milas.Influence of the method of purification on some solution properties of welan gum[J]Int.J.Biol.Macromol.1994 5(16) 253-257
[17]Tada,T.,Matsumoto,T..& Masuda,T..Structure of molecular association of curdlan at dilute regime in alkaline aqueous systems[J].Chem.Phys,1998,128-155
[18]Kim MK,Lee IY,Lee JH,et al.Residual phosphate concentration under nitrogen-limiting conditions regulates curdlan production in Agrobacterium sp.[J]Ind Microbiol.Biotechno 1.2000,25:180-183
[19]Kimura H,Sato S,Nakagawa T,et al.New thermo-geable polysaccharide containing foodstufs[P].US.Patent:3754925,1973-08-28
[20]陶金峰,原油价格为何居高难下.上海证券报网络版,2005
[21]Han D-K,Yang C-Z,Zhang Z-Q,et al.,Recent development of enhanced oil recovery in China[J].Journal of Petroleum Science and Engineering,1999,22(1):181-188
[22]Hoskin D H,Mitchell T O,Shu P.Oil reservoir permeability profile control with cross linked Welan gum biopolymers.U.S.Patent,4991652.1991
[23]李良雄,白宝君,李宇乡.油田深部驱剂的研究及应用[J].石油钻采工艺,1999,21(6):51-55
[24]Vercaemer C J,Davies S N,Pafitis D G,et al.,Selective zonal isolation of oil wells.U.S.Patent,5697441.1997
[25]詹晓北,韩杰.热凝胶的性质及其在食品中的应用[J],食品工业科技,2001,22(2):85-88
[26]Shlomo hestrint.The reaction of acetylcholine and other carboxylic acid derivatives with hydroxylamine and its analytical application[J].The journal of biological chemistry,1949,2(1)249-261
[27]张惟杰.复合多糖生化研究技术[M].上海:上海科技出版社,1987
[28]雷鸣,卢晓黎,陈正刚.黄原胶在低浓度时的流变特性及影响因素研究[J].食品科学,2000,21(12):16-18
[29]张俊,王军,陈碧兰.结冷胶水溶液的流变特性及其影响因素的研究[J].食品工业科技,2002,23(11):28-29
[30]牟世芬,刘克纳.离子色谱方法及应用[M].化学工业出版社,28-30
[31]杨仁智,张劲松,唐庆九.高效阴离子交换色谱法测定毛头鬼伞多糖中的单糖组成[J].中国食用菌,2005,24(5):42-44
[32]张培敏,朱岩,凌艳艳.离子色谱脉冲安培检测法测定水体颗粒物与河口沉积物中的糖[J].浙江大学学报,2004,31(4):431-434
[33]Kudoh Y,Shimizu J.,Ishiyama A.,et al.Secretion and excretion of immunoglobulin At ocecum and feces difer with type of indigestible saccharides[J].Journal of Nutritional Science and Vitaminology 1999,45(2):173-181:23
[34]Kanzawa.Y.,Koreeda,T.,Harada,A.,&Harada,T.Electron microscopy of the gel forming ability of polysaccharide food additives[J].Agric.Biel.Chem.1989a.53,979-986
[35]Chandrasekaran R;Radha A,Lee E J.Structural roles of calcium ions and side chains in welan:an x-ray study[J].Carbohydr.Res.,1994,252,183-207
[36]Polyelectrolyte and rheological studies on the polysaccharide welan[J]Int.J.Biol.Macromol.199012(12) 279-284
[37]徐佩弦.高聚物流变学及其应用,北京:化学工业出版社,2003
[38]陈芳,李建科,徐昶.新型微生物多糖-韦兰胶的流变特性影响因素研究[J]2007,28(9) 49-52
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