Klebsiella Oxytoca XCH-1菌的筛选及其胞外多糖的研究
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摘要
本文所研究的产酸克雷伯式菌(Klebsiella oxytoca XCH-1)是从青岛海藻化工厂海带浸泡液中筛。该菌在其生长过程中产生大量粘性的胞外多糖，为了进一步开发利用Klebsiella oxytoca XCH-1胞外多糖，本文对Klebsiella oxytoca XCH-1胞外多糖做了比较详细的研究。包括发酵，理化特性，结构及生物活性等。
本文采用发酵法制备Klebsiella oxytoca XCH-1胞外多糖。Klebsiella oxytoca
XCH-1菌发酵产生胞外多糖受多种因素的影响，如通气量、培养基的起始pH以及培养基的营养成分。实验结果表明，对于Klebsiella oxytoca XCH-1菌产胞外多糖的发酵，甘露醇为最佳碳源，硫酸铵为最佳氮源。采用均匀设计方法对发酵条件进行了研究，结论为甘露醇初始浓度为1％，C：N为50：1，温度为23℃，初始pH为8.18，接种量为9％。Klebsiella oxytoca XCH-1胞外多糖的产量为5.93mg／g。
首先发酵Klebsiella oxytoca XCH-1，并用乙醇沉淀的方法得到Klebsiella
oxytoca XCH-1胞外多糖粗品，利用Q—Sepharose FF阴离子交换树脂进行分级纯化，得到主要组分，并对其理化性质进行了全面的分析。Klebsiella oxytoca XCH-1胞外多糖为白色粉末，溶于水，并可溶于酸性、碱性溶液中。不溶于有机溶剂。Klebsiella oxytoca XCH-1胞外多糖的水溶液呈半透明粘稠状。用Sephaeryl S-300HR凝胶过滤色谱法测得分子量为156000左右。Klebsiella oxytoca XCH-1胞外多糖中硫酸基、蛋白质的含量为零。总糖含量为66.45％，糖醛酸含为15.89％。以0.1mol／L NaCl为溶剂配制0.5％的Klebsiella oxytoca XCH-1胞外多糖溶液，特性粘度为6.77。
Klebsiella oxytoca XCH-1胞外多糖具有较高的粘度，对热具有相当大的稳定性，短时间内，耐受100℃高温。pH4—8范围内，相对粘度基本不变。电解质对胞外多糖的相对粘度有一定的影响，随着NaCl、CaCl_2、Na_2B_4O_7浓度加大，相对粘度逐渐降低，当NaCl、CaCl_2、Na_2B_4O_7的浓度分别达到并超过3％、1％、1.5
尤才ebslella呷toca xcH一l菌的筛选及其胞外多糖的研究
%时，KlebsiellaO砂toca xcH一1胞外多糖溶液的相对粘度基本保持不变，表现出
一定的抗盐性。
犷飞本文研究了刀e五siella oxrtoca xeH一1胞外多糖对Pb(11)、ed(Ix)、er(vl)、
呵ll))的脱除能力。探讨了反应时间、重金属溶液浓度·溶液的pH值等因素对
丫少‘
，尹召bsiellao砂Ioca XCH一1胞外多糖脱除重金属能力的影响。结果表明Klebsiella
口习尸toca XCH一1胞外多糖对以上几种重金属离子有良好的脱除作用。刀e加i心lIa
口划toca XCH一1胞外多糖吸附重金属离子的能力顺序为F叹m)>Pb(n)>Cd(旦)
>cr(vi)。强酸性条件不利于KlebsiellaO划toca xcH一1胞外多糖对重金属离子豹
吸附，在弱酸性或接近中性时有利于吸附。
根据《食品安全性毒理学评价程序和方法》中急性经口毒性(LDs。)分级耘
准，通过小鼠急性经口毒性实验确定Klebsiella oxytoca xCH一1胞外多糖和磷貌
刀云bstellao大ytoca xCH一1胞外多糖可能为实际无毒物质。
本文将刀召bsiella口习尸toca XCH一1胞外多糖进行化学修饰，得到磺化刀‘bsi公fIa
。划犷口ca xcH一1胞外多糖，并用红外光谱验证了一05伪一取代的情况。对移蓦七
灯‘bsiellao荞Ftoca XCH一1胞外多糖的抗凝血活性进行了研究。实验表明，磺化
尤2云bsisllao砂toca xCH-1胞外多糖具有一定的抗凝血活性。
利用MTT法进行了淋巴细胞毒性实验、用ELIsA法测定了I刚一Y、IL一2、
IL一10、IL一12含量，实验表明刀‘bsiellao砂toca XCH一1胞外多糖及水解后豹
Klebsiella山卿toca xcH一1胞外多糖均有明显的免疫增强活性。有促进T、B细胸豹
增殖反应，虽然对T细胞直接活化所致的IFN一丫和IL一2的产生未见明显的影响葬
用，但对抗原提呈细胞活化所致的诱导IFN一Y产生有非常明显的增强作用。一
本文利用温和的酸水解得到低分子量的Klebsiella似叫oca xcH一1胞外多糖，
并对其进行了结构的分析，主要使用了高效凝胶过滤色谱、红外光谱、酸水解气
气相色谱、甲基化反应、气一质联用色谱和核磁共振等方法。
用HPSEC法测定其分子量为40200左右。用GC分析低分子刀‘bsiella
XCH一1胞外多糖，其主要单糖组成为鼠李糖、半乳糖、葡萄糖。利用核磁共振普
谱法对低分子的人介bsiella oxytoca xcH一1胞外多糖进行结构分析，结合甲基化豹
Klebsiellaa习户toca xcH一l菌的筛选及其胞外多糖的研究
结果，证明主链结构中:鼠李糖有a、p两种构型，葡萄糖主要有p构型，半乳
糖主要有Q构型。支链葡萄糖与主链鼠李糖以1一2连接，其构型也为p构型。
The bacterium Klebsiella oxytoca XCH-1 strain studied in this thesis was isolated and obtained from the water extract of Laminaria Japonica in Qingdao Algae Chemical Plant. It produces a large quantity of viscous exopolysaccharide while growing. For further study and exploitation of the exopolysaccharide, intensive analyses on it have been made and depicted in this paper.
In this study, exopolysaccharide was prepared by fermentation of K .oxytoca XCH-1 strain.Many factors, such as, aeration, initial pH of cultivating substrate and nutritional composition of the substrate, can affect the yield of exopolysaccharide by fermentation of K. oxytoca XCH-1 strain. Experiment results show that as to fermentation of AT. oxytoca XCH-1 strain for producing exopolysaccharide, mannite is the best carbon source, and (NH4)SO4 is the best nitrogen source. Fermentation conditions have been studied by Uniform Design Test. The conclusion is that the yield of exopolysaccharide from K. oxytoca XCH-1 is 5.93 mg/g on the condition of initial mannite concentration as 1%, C : N as 50 : 1, temperature as 23 C, initial pH value as 8.18, inoculation quantum as 9%.
After fermentation of K.oxytoca XCH-1 strain, crude exopolysaccharide was then obtained by precipitating it in ethanol. The crude product was purified in gradation by anion-exchange ( Q-Sepharose FF ), thus the main fraction of it was obtained. The purified product was thoroughly analyzed for its physical and chemical properties. The exopolysaccharide is a white powder, soluble in water, and can be dissolved into acid or alkaline solution, but shows no solubility in organic solvent. The exopolysaccharide water solution is viscous and translucent. Its average molecular weight is examined as 156000 or so by gel filtration chromatography with Sephaeryl S-300HR column. The exopolysaccharide is free from sulphate group and protein, and with 66.45% of total carbohydrates, 15.89% of uronic acids, so it presents acidic polyssacharide characteristic. With 0.1mol/l of NaCl solution as solvent, 0.5% of the exopolysaccharide solution was prepared, its intrinsic viscosity tested as 6.77 at 25C. The exopolysaccharide is o
f high viscosity and of significant toleraice to heat. It can stand the temperature of 100C for a short time. The relative viscosity is constant in the range of pH 4-8. Electrolytes have impact on the exopolysaccharide to some extent. The relative viscosity of the exopolysaccharide solution is declining as concentration of NaCl or CaCl2 or Na2B4O7 increases. When concentration of the above electrolytes respectively exceed 3%, 1%, 1.5%, the relative viscosity of the exopolysaccharide solution basically keep unchanged, hence shows some tolerance to electrolytes.
In this thesis, removal effect of the exopolysaccharide for lead (PbII), cadmium (CdII), chromium (CrVI), iron (FeIII), as well as the effect of reaction time, concentrations of
heavy metal solutions, pH value of the solutions on its removal capability for these heavy metals have been studied.The results show excellent removal effect of the exopolysaccharide for those heavy metals. The sequence from high to low for capability of binding heavy metals of the exopolysaccharide is Fe(III)>Pb(II)> Cd(II)>Cr(VI). Weak acid or neutral condition is more favorable for heavy metal binding than strong acid condition.
According to graded standards of acute oral toxicity ( LD50 ) regulated by "Toxicological evaluation procedure and method for food safety", the exopolysaccharide and its sulphatised products were tested of no toxicity by acute oral toxicity test in mice and they are most probably toxic free substance. The exopolysaccharide in this study was modified by sulphatizing and the sulphatised exopolysaccharide was then tested for the successful substitution of -OSO3- by infrared spectroscopy and also studied for its anticoagulant activity. The experiment reveals that the sulphatised exopolysaccharide have significant anticoagulant activity. The acute toxicological test for lymphocyte was carried out by MTT method, aad conte
本文采用发酵法制备Klebsiella oxytoca XCH-1胞外多糖。Klebsiella oxytoca
XCH-1菌发酵产生胞外多糖受多种因素的影响，如通气量、培养基的起始pH以及培养基的营养成分。实验结果表明，对于Klebsiella oxytoca XCH-1菌产胞外多糖的发酵，甘露醇为最佳碳源，硫酸铵为最佳氮源。采用均匀设计方法对发酵条件进行了研究，结论为甘露醇初始浓度为1％，C：N为50：1，温度为23℃，初始pH为8.18，接种量为9％。Klebsiella oxytoca XCH-1胞外多糖的产量为5.93mg／g。
首先发酵Klebsiella oxytoca XCH-1，并用乙醇沉淀的方法得到Klebsiella
oxytoca XCH-1胞外多糖粗品，利用Q—Sepharose FF阴离子交换树脂进行分级纯化，得到主要组分，并对其理化性质进行了全面的分析。Klebsiella oxytoca XCH-1胞外多糖为白色粉末，溶于水，并可溶于酸性、碱性溶液中。不溶于有机溶剂。Klebsiella oxytoca XCH-1胞外多糖的水溶液呈半透明粘稠状。用Sephaeryl S-300HR凝胶过滤色谱法测得分子量为156000左右。Klebsiella oxytoca XCH-1胞外多糖中硫酸基、蛋白质的含量为零。总糖含量为66.45％，糖醛酸含为15.89％。以0.1mol／L NaCl为溶剂配制0.5％的Klebsiella oxytoca XCH-1胞外多糖溶液，特性粘度为6.77。
Klebsiella oxytoca XCH-1胞外多糖具有较高的粘度，对热具有相当大的稳定性，短时间内，耐受100℃高温。pH4—8范围内，相对粘度基本不变。电解质对胞外多糖的相对粘度有一定的影响，随着NaCl、CaCl_2、Na_2B_4O_7浓度加大，相对粘度逐渐降低，当NaCl、CaCl_2、Na_2B_4O_7的浓度分别达到并超过3％、1％、1.5
尤才ebslella呷toca xcH一l菌的筛选及其胞外多糖的研究
%时，KlebsiellaO砂toca xcH一1胞外多糖溶液的相对粘度基本保持不变，表现出
一定的抗盐性。
犷飞本文研究了刀e五siella oxrtoca xeH一1胞外多糖对Pb(11)、ed(Ix)、er(vl)、
呵ll))的脱除能力。探讨了反应时间、重金属溶液浓度·溶液的pH值等因素对
丫少‘
，尹召bsiellao砂Ioca XCH一1胞外多糖脱除重金属能力的影响。结果表明Klebsiella
口习尸toca XCH一1胞外多糖对以上几种重金属离子有良好的脱除作用。刀e加i心lIa
口划toca XCH一1胞外多糖吸附重金属离子的能力顺序为F叹m)>Pb(n)>Cd(旦)
>cr(vi)。强酸性条件不利于KlebsiellaO划toca xcH一1胞外多糖对重金属离子豹
吸附，在弱酸性或接近中性时有利于吸附。
根据《食品安全性毒理学评价程序和方法》中急性经口毒性(LDs。)分级耘
准，通过小鼠急性经口毒性实验确定Klebsiella oxytoca xCH一1胞外多糖和磷貌
刀云bstellao大ytoca xCH一1胞外多糖可能为实际无毒物质。
本文将刀召bsiella口习尸toca XCH一1胞外多糖进行化学修饰，得到磺化刀‘bsi公fIa
。划犷口ca xcH一1胞外多糖，并用红外光谱验证了一05伪一取代的情况。对移蓦七
灯‘bsiellao荞Ftoca XCH一1胞外多糖的抗凝血活性进行了研究。实验表明，磺化
尤2云bsisllao砂toca xCH-1胞外多糖具有一定的抗凝血活性。
利用MTT法进行了淋巴细胞毒性实验、用ELIsA法测定了I刚一Y、IL一2、
IL一10、IL一12含量，实验表明刀‘bsiellao砂toca XCH一1胞外多糖及水解后豹
Klebsiella山卿toca xcH一1胞外多糖均有明显的免疫增强活性。有促进T、B细胸豹
增殖反应，虽然对T细胞直接活化所致的IFN一丫和IL一2的产生未见明显的影响葬
用，但对抗原提呈细胞活化所致的诱导IFN一Y产生有非常明显的增强作用。一
本文利用温和的酸水解得到低分子量的Klebsiella似叫oca xcH一1胞外多糖，
并对其进行了结构的分析，主要使用了高效凝胶过滤色谱、红外光谱、酸水解气
气相色谱、甲基化反应、气一质联用色谱和核磁共振等方法。
用HPSEC法测定其分子量为40200左右。用GC分析低分子刀‘bsiella
XCH一1胞外多糖，其主要单糖组成为鼠李糖、半乳糖、葡萄糖。利用核磁共振普
谱法对低分子的人介bsiella oxytoca xcH一1胞外多糖进行结构分析，结合甲基化豹
Klebsiellaa习户toca xcH一l菌的筛选及其胞外多糖的研究
结果，证明主链结构中:鼠李糖有a、p两种构型，葡萄糖主要有p构型，半乳
糖主要有Q构型。支链葡萄糖与主链鼠李糖以1一2连接，其构型也为p构型。
The bacterium Klebsiella oxytoca XCH-1 strain studied in this thesis was isolated and obtained from the water extract of Laminaria Japonica in Qingdao Algae Chemical Plant. It produces a large quantity of viscous exopolysaccharide while growing. For further study and exploitation of the exopolysaccharide, intensive analyses on it have been made and depicted in this paper.
In this study, exopolysaccharide was prepared by fermentation of K .oxytoca XCH-1 strain.Many factors, such as, aeration, initial pH of cultivating substrate and nutritional composition of the substrate, can affect the yield of exopolysaccharide by fermentation of K. oxytoca XCH-1 strain. Experiment results show that as to fermentation of AT. oxytoca XCH-1 strain for producing exopolysaccharide, mannite is the best carbon source, and (NH4)SO4 is the best nitrogen source. Fermentation conditions have been studied by Uniform Design Test. The conclusion is that the yield of exopolysaccharide from K. oxytoca XCH-1 is 5.93 mg/g on the condition of initial mannite concentration as 1%, C : N as 50 : 1, temperature as 23 C, initial pH value as 8.18, inoculation quantum as 9%.
After fermentation of K.oxytoca XCH-1 strain, crude exopolysaccharide was then obtained by precipitating it in ethanol. The crude product was purified in gradation by anion-exchange ( Q-Sepharose FF ), thus the main fraction of it was obtained. The purified product was thoroughly analyzed for its physical and chemical properties. The exopolysaccharide is a white powder, soluble in water, and can be dissolved into acid or alkaline solution, but shows no solubility in organic solvent. The exopolysaccharide water solution is viscous and translucent. Its average molecular weight is examined as 156000 or so by gel filtration chromatography with Sephaeryl S-300HR column. The exopolysaccharide is free from sulphate group and protein, and with 66.45% of total carbohydrates, 15.89% of uronic acids, so it presents acidic polyssacharide characteristic. With 0.1mol/l of NaCl solution as solvent, 0.5% of the exopolysaccharide solution was prepared, its intrinsic viscosity tested as 6.77 at 25C. The exopolysaccharide is o
f high viscosity and of significant toleraice to heat. It can stand the temperature of 100C for a short time. The relative viscosity is constant in the range of pH 4-8. Electrolytes have impact on the exopolysaccharide to some extent. The relative viscosity of the exopolysaccharide solution is declining as concentration of NaCl or CaCl2 or Na2B4O7 increases. When concentration of the above electrolytes respectively exceed 3%, 1%, 1.5%, the relative viscosity of the exopolysaccharide solution basically keep unchanged, hence shows some tolerance to electrolytes.
In this thesis, removal effect of the exopolysaccharide for lead (PbII), cadmium (CdII), chromium (CrVI), iron (FeIII), as well as the effect of reaction time, concentrations of
heavy metal solutions, pH value of the solutions on its removal capability for these heavy metals have been studied.The results show excellent removal effect of the exopolysaccharide for those heavy metals. The sequence from high to low for capability of binding heavy metals of the exopolysaccharide is Fe(III)>Pb(II)> Cd(II)>Cr(VI). Weak acid or neutral condition is more favorable for heavy metal binding than strong acid condition.
According to graded standards of acute oral toxicity ( LD50 ) regulated by "Toxicological evaluation procedure and method for food safety", the exopolysaccharide and its sulphatised products were tested of no toxicity by acute oral toxicity test in mice and they are most probably toxic free substance. The exopolysaccharide in this study was modified by sulphatizing and the sulphatised exopolysaccharide was then tested for the successful substitution of -OSO3- by infrared spectroscopy and also studied for its anticoagulant activity. The experiment reveals that the sulphatised exopolysaccharide have significant anticoagulant activity. The acute toxicological test for lymphocyte was carried out by MTT method, aad conte
引文
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