紫外诱变纤维素分解菌降解木薯渣的研究
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摘要
木薯渣易造成环境污染,加之粗纤维含量过多,不利于动物消化利用。为了解决这一问题,通过微生物降解木薯渣,使其纤维素含量降低、还原糖含量增高,并对混菌发酵产菌体蛋白进行了初步探讨。
本研究以实验室保藏的黑曲霉作为原始菌株,通过在纤维素培养基上富集培养,经紫外线辐射诱变,确定最适诱变条件为:用30 W紫外灯,垂直距离30 cm,辐射时间为3 min,得到一株高产纤维素酶的突变菌株GL-1,其降解纤维素产还原糖量403.57 mg/g,为出发菌株的1.33倍。
为了进一步提高目的菌株对木薯渣的降解率,增加其还原糖含量,对黑曲霉GL-1发酵工艺进行研究。采用Plackett-Burman试验筛选对发酵影响显著的因素:固态发酵为初始pH、温度、培养基含水量;液态发酵为pH、发酵温度、含氮量。分别对其进行单因素试验以确定适当的水平。固态发酵采用响应面试验(中心点试验N = 5)确定最优工艺条件为:初始pH 7.5、温度35℃、培养基含水量4 mL/g;液态发酵采用L9(33)正交试验,确定最优工艺条件为:pH 8、发酵温度30℃、含氮量0.5%。最优条件下各发酵3 d,测得其还原糖含量分别为435.7 mg/g和395.6 mg/mL,对比试验结果可知固态发酵较液态发酵效果好。
发酵所产还原糖提纯后通过高效液相色谱分析得出其中含有2.61%鼠李糖、50.28%葡萄糖、25.37%果糖、2.57%蔗糖和1.85%麦芽糖。
对固态发酵产物的常规分析表明木薯渣经黑曲霉GL-1降解后,其粗纤维含量变化影响很大,与未发酵木薯渣相比其中粗纤维含量由15.46%降至8.47%。
为提高发酵产物蛋白含量,研究了黑曲霉GL-1与酵母菌的拮抗关系,结果表明两种菌之间不存在拮抗。混菌接种量为7%～12%,25℃～35℃固态发酵4 d,考马斯亮蓝法测其蛋白含量为12.18%,相比原料提高了10.25%。
混菌发酵产物进行氰化物定性检测,未检出氰化物。
Cassava residue can easily lead to environmental pollution, it is not conducive animal digestion for its too much crude fiber content. In order to solve the problm, the biological degradation on cassava residue is used, and the result of it is the decrease of cellulose content and the increase of reducing sugar content. And the production of cell protein by mixed fermentation was simply discussed in this study.
Aspergillus niger was selected as the initial strain for breeding cellulase-producing mutants. A mutant strain, GL-1 was obtained through mutagenization with ultraviolet(UV). The optimal mutation conditions were illumination with 30 W UV lamp for 3 minutes at a vertical distance of 30 cm. The reducing sugar content of this high-yield strain reached 403.57 mg/g, which was 1.33 times of that of initial strain.
To further enhance the objective strain on the degradation of cassava and increase the content of its reducing sugar, the fermentation conditions of Aspergillus niger GL-1 were studied. And through Plackett-Burman single-factor test screening significant factors on the fermentation: solid state fermentation are the initial pH of fermentation, fermentation temperature, the content of water, liquid state fermentation are pH, fermentation temperature, the content of nitrogen. The appropriate level was determined by single-factor test. Solid state fermentation, via the response surface test, the optimum fermentation conditions were as follows: the initial pH 6.52, 35℃, water content 4 mL/g; Solid state fermentation, through orthogonal experiment, the optimum fermentation conditions were as follows: the initial pH was 8, the fermentation temperature was 30℃, the content of nitrogen was 0.5%. Under optimal conditions the fermentation was 3 d. The content of reducing sugar were 435.7 mg/g and 395.6 mg/mL, respectly. The results of experiment showed that solid state fermentation was better than liquid state fermentation.
The result of HPLC analysis of oligosaccharide composition.The fermentation product includes 2.61% rhamnose, 50.28% glucose, 25.37% fructose, 2.57% sucrose and 1.85% maltose.
The conventional analysis to product of solid state fermentation showed that the dual fermentation create a great change of crude fiber content in cassava residue. And compared to non-fermented cassava residue, the fermentation product decrease crude fiber from 15.46 percent to 8.47 percent.
The antagonistic test showed that there was no antagonism between the Aspergillus niger GL-1 and the yeast. Mixed bacterial inoculum was 7%～12%, 25℃～35℃solid-state fermentated 4 d, then the protein content was 12.18 % which was tested by coomassie brilliant blue method, increased than raw materials by 10.25%.
Through the cyanide test, there was no cyanide in the fermentation product.
本研究以实验室保藏的黑曲霉作为原始菌株,通过在纤维素培养基上富集培养,经紫外线辐射诱变,确定最适诱变条件为:用30 W紫外灯,垂直距离30 cm,辐射时间为3 min,得到一株高产纤维素酶的突变菌株GL-1,其降解纤维素产还原糖量403.57 mg/g,为出发菌株的1.33倍。
为了进一步提高目的菌株对木薯渣的降解率,增加其还原糖含量,对黑曲霉GL-1发酵工艺进行研究。采用Plackett-Burman试验筛选对发酵影响显著的因素:固态发酵为初始pH、温度、培养基含水量;液态发酵为pH、发酵温度、含氮量。分别对其进行单因素试验以确定适当的水平。固态发酵采用响应面试验(中心点试验N = 5)确定最优工艺条件为:初始pH 7.5、温度35℃、培养基含水量4 mL/g;液态发酵采用L9(33)正交试验,确定最优工艺条件为:pH 8、发酵温度30℃、含氮量0.5%。最优条件下各发酵3 d,测得其还原糖含量分别为435.7 mg/g和395.6 mg/mL,对比试验结果可知固态发酵较液态发酵效果好。
发酵所产还原糖提纯后通过高效液相色谱分析得出其中含有2.61%鼠李糖、50.28%葡萄糖、25.37%果糖、2.57%蔗糖和1.85%麦芽糖。
对固态发酵产物的常规分析表明木薯渣经黑曲霉GL-1降解后,其粗纤维含量变化影响很大,与未发酵木薯渣相比其中粗纤维含量由15.46%降至8.47%。
为提高发酵产物蛋白含量,研究了黑曲霉GL-1与酵母菌的拮抗关系,结果表明两种菌之间不存在拮抗。混菌接种量为7%～12%,25℃～35℃固态发酵4 d,考马斯亮蓝法测其蛋白含量为12.18%,相比原料提高了10.25%。
混菌发酵产物进行氰化物定性检测,未检出氰化物。
Cassava residue can easily lead to environmental pollution, it is not conducive animal digestion for its too much crude fiber content. In order to solve the problm, the biological degradation on cassava residue is used, and the result of it is the decrease of cellulose content and the increase of reducing sugar content. And the production of cell protein by mixed fermentation was simply discussed in this study.
Aspergillus niger was selected as the initial strain for breeding cellulase-producing mutants. A mutant strain, GL-1 was obtained through mutagenization with ultraviolet(UV). The optimal mutation conditions were illumination with 30 W UV lamp for 3 minutes at a vertical distance of 30 cm. The reducing sugar content of this high-yield strain reached 403.57 mg/g, which was 1.33 times of that of initial strain.
To further enhance the objective strain on the degradation of cassava and increase the content of its reducing sugar, the fermentation conditions of Aspergillus niger GL-1 were studied. And through Plackett-Burman single-factor test screening significant factors on the fermentation: solid state fermentation are the initial pH of fermentation, fermentation temperature, the content of water, liquid state fermentation are pH, fermentation temperature, the content of nitrogen. The appropriate level was determined by single-factor test. Solid state fermentation, via the response surface test, the optimum fermentation conditions were as follows: the initial pH 6.52, 35℃, water content 4 mL/g; Solid state fermentation, through orthogonal experiment, the optimum fermentation conditions were as follows: the initial pH was 8, the fermentation temperature was 30℃, the content of nitrogen was 0.5%. Under optimal conditions the fermentation was 3 d. The content of reducing sugar were 435.7 mg/g and 395.6 mg/mL, respectly. The results of experiment showed that solid state fermentation was better than liquid state fermentation.
The result of HPLC analysis of oligosaccharide composition.The fermentation product includes 2.61% rhamnose, 50.28% glucose, 25.37% fructose, 2.57% sucrose and 1.85% maltose.
The conventional analysis to product of solid state fermentation showed that the dual fermentation create a great change of crude fiber content in cassava residue. And compared to non-fermented cassava residue, the fermentation product decrease crude fiber from 15.46 percent to 8.47 percent.
The antagonistic test showed that there was no antagonism between the Aspergillus niger GL-1 and the yeast. Mixed bacterial inoculum was 7%～12%, 25℃～35℃solid-state fermentated 4 d, then the protein content was 12.18 % which was tested by coomassie brilliant blue method, increased than raw materials by 10.25%.
Through the cyanide test, there was no cyanide in the fermentation product.
引文
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