假单胞菌利用玉米芯水解液产胞外多糖研究
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摘要
本研究主要目的是提高假单胞菌胞利用玉米芯水解液生产胞外多糖产量，通过优化发酵条件和对产生菌诱变育种为主要手段实现。
在假单胞菌基本特征研究中发现，实验菌株易于利用葡萄糖、木糖和蔗糖生长，并产多糖。培养基中碳源、氮源浓度和碳氮比对多糖的产生起关键作用，碳氮比低于3时已没有多余的碳源用于次生代谢产物胞外多糖的合成。在低浓度无机氮或有机氮的情况下，多糖产量和粘度都明显比高浓度氮源时要高。在提高发酵液粘度方面有机氮源略有优势，但对多糖产量没有明显影响。通过实验确定在培养基中添加0.02%氯化铵作为生长限制因子以提高多糖产量。
在高碳氮比培养条件下，假单胞菌生长达到稳定期时，OD值仍有快速增加的趋势，说明实验菌株是在稳定期开始分泌胞外多糖。在固体培养时产多糖菌落为同心圆形，外围透明圈为多糖，可作为诱变育种时的初筛指标。
比较了不同玉米芯粗制品对发酵的影响，发现粉碎后过2.0目筛下细料、筛上粗料与未分筛的混合料对发酵效果的影响为：细料＞混合料＞粗料，但差异并不大，尤其前两者之间。考虑加工工艺、生产成本及对原料的充分利用等因素，因此确定直接采用粉碎后的玉米芯粉作为生产原料。实验还发现原料加倍时多糖产量也相应加倍，最高可达26.96g/L。
一些对黄原胶发酵有促进作用的因子对实验菌株产多糖没有影响。而一定浓度的微量元素对多糖产量有促生作用，最后确定微量元素溶液的配方为1000ml母液中添加MnSO4 0.3克，CaCl2 0.2克，FeSO4 0.8克。
实验菌株合成多糖时需要高通气量；培养液起始pH值在7.5～10.0之间均能生长产糖，但不能低于7.0，最适起始pH为8.0；在28～32℃都可以生产产糖；加大接种量可缩短发酵时间；5天时产量已经达到最高。最后确定采用30小时菌龄，5%接种量，20%装液量为最高，初始pH值为8.0，30℃/180rpm振荡培养5天为最佳发酵条件。
为了提高多糖产量，以低能N+注入和紫外线辐射作为诱变手段对实验菌株进行了诱变育种。研究发现紫外线杀菌曲线呈现常规性“肩型”曲线；而采用能量为10KevN+注入处理时，假单胞菌存活率出现先降后升而后又降的特征性“马鞍型”曲线。通过紫外线处理获得高产菌株UVZT122、UVZT213，其产量分别提高了10.53%和9.57%；通过低能N+注入处理，获得高产菌株NZTZ07和NZTZ11，其产量分别提高了11.02%和6.83%。
假单胞菌多糖溶液流变学性质研究发现，该多糖溶液具有自身增粘性和触变性，
属于假塑性流体。多糖溶液具有很强的环境适应性：pH值在1～14之间粘度没有明显影响；耐高温，在60℃保持20分钟粘度没有变化；添加NaCl、CaCO3等无机盐对粘度不产生影响。Fe3+对该多糖溶液具有增粘性，当多糖与Fe2(SO4)3质量比为2时形成凝胶。强酸、强碱对多糖水解效果依次为HCl＞ NaOH＞ H2SO4，添加NaC可使多糖溶液在煮沸状况下保持粘度不变。
In this study, the main object is to enhance exopolysaccharide product using corncob as carbon source. In order to do this, the conditions of fermentation of exopolysaccharide by Pseudomonas were researched, and the strain was treated by ultraviolet and low-energy nitrogen ion beam implantation.
This strain could utilize glucose, xylose and sucrose to produce exopolysaccharide. Carbon source concentration, nitrogen source concentration and carbon-nitrogen ratio are key roles to produce exopolysaccharide. If carbon-nitrogen ratio was below 3, there was no carbon source to be used to produce exopolysaccharide. As for nitrogen source, low concentration is better than high concentration. Organic nitrogen is prior to inorganic one to get high viscidity, but it had no effect on yield. At last 0.02% ammonium chloride was chose as limited factor to enhance exopolysaccharide product.
OD value was still ascending after the stationary phase when medium contain high concentration glucose, and this shows that Pseudomonas began to produce exopolysaccharide in the stationary phase. The colony was concentric circles shaping in solid medium. Exopolysaccharide formed transparent ring outside of colony, which can be used to screen mutants having high yield of exopolysaccharide.
In consideration of process of corncob, yielding cost and full using etc, mixing stuff of corncob was chosen, which the distinguishing size is 2.0-eye griddle hole. Meanwhile, exopolysaccharide productivity was double if two-dose material was used in experiment. The yield of exopolysaccharide was 26.96g/L.
Calcium carbonate, tween 80 and lemon acid had no effect on polysaccharide production referring to xanthan study. But trace elements could facilitate the yield of exopolysaccharide. The new prescription of trace element solution (g/1000ml) was as following: MnSO4 0.300，CaCl2 0.2，FeSO4 0.8.
For producing exopolysaccharide, high air concentration was needed. Pseudomonas sp could grow and produce exopolysaccharide in pH range from 7.5 to 10.0, but could not below pH7.0, and the optimum initial pH is 8.0. Meanwhile, it could grow and produce exopolysaccharide at 28～32℃, but could not below 26 or higher than 35℃. Amount of inoculation had no effect on fermentation, but higher amount of inoculation could shorten producing period. The fermentation period was set five days because of the yield is much higher at this time. The yield was steadiness at day 5, but viscosity still enlarged
continually probably because of water evaporation. The optimal fermentation condition was 30h bacterial age, 5% inoculating amount, no more than 20% load, initial pH 8.0, 30℃/180rpm and five days growth.
Low energy nitrogen ion beam implantation and ultraviolet light were chosen as mutagens to treat strains. As the result, the UV-bactericidal-curve appearing a routine “shoulder shape”. While 10KevN+ radiation was introduced, the survive ratio decrease first, then increase and drop at last. The curve was “saddle shape”. Finally, two high yield strains, UVZT122 and UVZT213 were got, the yield of which was enhanced by 10.53% and 9.57% respectively. And two high yield strains, NZTZ07 and NZTZ11 were got by low energy N ion beam implantation, the yield of which was enhanced by 11.02% and 6.83% respectively.
Rheology research found that the exopolysaccharide had self-viscosity-improvement and thixotropy, so the exopolysaccharide solution was a pseudoplastic liquid. The exopolysaccharide had good adaptability to environment conditions. The viscidity had no change in the range of pH 1～14, and at 60℃. It could be untouched for 20 minutes. And so did it when NaCl, CaCO3 was added. Additionally, Fe3+ could enhance viscidity of the exopolysaccharide solution. When the ratio of the exopolysaccharide and Fe2(SO4) 3 was 2, the solution of exopolysaccharide became a gel. The hydraylation of exopolysaccharide by acid or base was: HCl＞ NaOH＞ H2SO4. It prevented the viscidity decrease from heating by adding NaCl.
在假单胞菌基本特征研究中发现，实验菌株易于利用葡萄糖、木糖和蔗糖生长，并产多糖。培养基中碳源、氮源浓度和碳氮比对多糖的产生起关键作用，碳氮比低于3时已没有多余的碳源用于次生代谢产物胞外多糖的合成。在低浓度无机氮或有机氮的情况下，多糖产量和粘度都明显比高浓度氮源时要高。在提高发酵液粘度方面有机氮源略有优势，但对多糖产量没有明显影响。通过实验确定在培养基中添加0.02%氯化铵作为生长限制因子以提高多糖产量。
在高碳氮比培养条件下，假单胞菌生长达到稳定期时，OD值仍有快速增加的趋势，说明实验菌株是在稳定期开始分泌胞外多糖。在固体培养时产多糖菌落为同心圆形，外围透明圈为多糖，可作为诱变育种时的初筛指标。
比较了不同玉米芯粗制品对发酵的影响，发现粉碎后过2.0目筛下细料、筛上粗料与未分筛的混合料对发酵效果的影响为：细料＞混合料＞粗料，但差异并不大，尤其前两者之间。考虑加工工艺、生产成本及对原料的充分利用等因素，因此确定直接采用粉碎后的玉米芯粉作为生产原料。实验还发现原料加倍时多糖产量也相应加倍，最高可达26.96g/L。
一些对黄原胶发酵有促进作用的因子对实验菌株产多糖没有影响。而一定浓度的微量元素对多糖产量有促生作用，最后确定微量元素溶液的配方为1000ml母液中添加MnSO4 0.3克，CaCl2 0.2克，FeSO4 0.8克。
实验菌株合成多糖时需要高通气量；培养液起始pH值在7.5～10.0之间均能生长产糖，但不能低于7.0，最适起始pH为8.0；在28～32℃都可以生产产糖；加大接种量可缩短发酵时间；5天时产量已经达到最高。最后确定采用30小时菌龄，5%接种量，20%装液量为最高，初始pH值为8.0，30℃/180rpm振荡培养5天为最佳发酵条件。
为了提高多糖产量，以低能N+注入和紫外线辐射作为诱变手段对实验菌株进行了诱变育种。研究发现紫外线杀菌曲线呈现常规性“肩型”曲线；而采用能量为10KevN+注入处理时，假单胞菌存活率出现先降后升而后又降的特征性“马鞍型”曲线。通过紫外线处理获得高产菌株UVZT122、UVZT213，其产量分别提高了10.53%和9.57%；通过低能N+注入处理，获得高产菌株NZTZ07和NZTZ11，其产量分别提高了11.02%和6.83%。
假单胞菌多糖溶液流变学性质研究发现，该多糖溶液具有自身增粘性和触变性，
属于假塑性流体。多糖溶液具有很强的环境适应性：pH值在1～14之间粘度没有明显影响；耐高温，在60℃保持20分钟粘度没有变化；添加NaCl、CaCO3等无机盐对粘度不产生影响。Fe3+对该多糖溶液具有增粘性，当多糖与Fe2(SO4)3质量比为2时形成凝胶。强酸、强碱对多糖水解效果依次为HCl＞ NaOH＞ H2SO4，添加NaC可使多糖溶液在煮沸状况下保持粘度不变。
In this study, the main object is to enhance exopolysaccharide product using corncob as carbon source. In order to do this, the conditions of fermentation of exopolysaccharide by Pseudomonas were researched, and the strain was treated by ultraviolet and low-energy nitrogen ion beam implantation.
This strain could utilize glucose, xylose and sucrose to produce exopolysaccharide. Carbon source concentration, nitrogen source concentration and carbon-nitrogen ratio are key roles to produce exopolysaccharide. If carbon-nitrogen ratio was below 3, there was no carbon source to be used to produce exopolysaccharide. As for nitrogen source, low concentration is better than high concentration. Organic nitrogen is prior to inorganic one to get high viscidity, but it had no effect on yield. At last 0.02% ammonium chloride was chose as limited factor to enhance exopolysaccharide product.
OD value was still ascending after the stationary phase when medium contain high concentration glucose, and this shows that Pseudomonas began to produce exopolysaccharide in the stationary phase. The colony was concentric circles shaping in solid medium. Exopolysaccharide formed transparent ring outside of colony, which can be used to screen mutants having high yield of exopolysaccharide.
In consideration of process of corncob, yielding cost and full using etc, mixing stuff of corncob was chosen, which the distinguishing size is 2.0-eye griddle hole. Meanwhile, exopolysaccharide productivity was double if two-dose material was used in experiment. The yield of exopolysaccharide was 26.96g/L.
Calcium carbonate, tween 80 and lemon acid had no effect on polysaccharide production referring to xanthan study. But trace elements could facilitate the yield of exopolysaccharide. The new prescription of trace element solution (g/1000ml) was as following: MnSO4 0.300，CaCl2 0.2，FeSO4 0.8.
For producing exopolysaccharide, high air concentration was needed. Pseudomonas sp could grow and produce exopolysaccharide in pH range from 7.5 to 10.0, but could not below pH7.0, and the optimum initial pH is 8.0. Meanwhile, it could grow and produce exopolysaccharide at 28～32℃, but could not below 26 or higher than 35℃. Amount of inoculation had no effect on fermentation, but higher amount of inoculation could shorten producing period. The fermentation period was set five days because of the yield is much higher at this time. The yield was steadiness at day 5, but viscosity still enlarged
continually probably because of water evaporation. The optimal fermentation condition was 30h bacterial age, 5% inoculating amount, no more than 20% load, initial pH 8.0, 30℃/180rpm and five days growth.
Low energy nitrogen ion beam implantation and ultraviolet light were chosen as mutagens to treat strains. As the result, the UV-bactericidal-curve appearing a routine “shoulder shape”. While 10KevN+ radiation was introduced, the survive ratio decrease first, then increase and drop at last. The curve was “saddle shape”. Finally, two high yield strains, UVZT122 and UVZT213 were got, the yield of which was enhanced by 10.53% and 9.57% respectively. And two high yield strains, NZTZ07 and NZTZ11 were got by low energy N ion beam implantation, the yield of which was enhanced by 11.02% and 6.83% respectively.
Rheology research found that the exopolysaccharide had self-viscosity-improvement and thixotropy, so the exopolysaccharide solution was a pseudoplastic liquid. The exopolysaccharide had good adaptability to environment conditions. The viscidity had no change in the range of pH 1～14, and at 60℃. It could be untouched for 20 minutes. And so did it when NaCl, CaCO3 was added. Additionally, Fe3+ could enhance viscidity of the exopolysaccharide solution. When the ratio of the exopolysaccharide and Fe2(SO4) 3 was 2, the solution of exopolysaccharide became a gel. The hydraylation of exopolysaccharide by acid or base was: HCl＞ NaOH＞ H2SO4. It prevented the viscidity decrease from heating by adding NaCl.
引文
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