黑曲霉（Aspergillus niger sp.）固态发酵啤酒糟生产纤维素酶及其酶学性质与发酵产物的研究
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摘要
啤酒糟是啤酒工业中的主要副产物之一。目前,大多数厂家将其当饲料出售,有的甚至当废物排放,污染环境的同时也造成了资源的浪费。啤酒糟的主要成分是麦芽壳和未糖化的麦芽组分,其中含有大量的纤维素,而纤维素是纤维素酶的诱导物,而且啤酒糟中含有一定量的含氮化合物和多种无机元素及维生素,质地疏松,是固态发酵生产纤维素酶的优良基质。本研究以啤酒糟为主要原料,对黑曲霉(Aspergillus niger sp.)固态发酵生产纤维素酶的发酵工艺条件、酶学性质以及发酵对培养基组成的影响进行了研究,主要研究结果如下:
1.通过单因素实验对黑曲霉发酵啤酒糟生产纤维素酶的发酵工艺条件进行了初步研究,适宜的培养基组分为:500 mL三角瓶中装入啤酒糟和棉粕20 g,配料比8:2,料水比1:1.5,于30℃发酵66 h,滤纸酶活(FPA)和羧甲基纤维素酶活(CMCase)分别达到759.9±51.7 U/g和14187.8±579.1 U/g(干物质);在含氮量相等的条件下,试验所用的(NH4)2SO4、NH4Cl、NaNO3、(NH2)2CO、(NH4)2HPO4对酶活影响不显著;KH2PO4和CaCl2分别在0～2.5%和0～1.6%的添加范围内对产酶影响也不显著。
2.在单因素研究的基础上,采用三因素二次通用旋转组合试验,研究了啤酒糟与棉粕的配料比、料水比和发酵时间对黑曲霉固态发酵啤酒糟生产纤维素酶的影响,建立了纤维素酶随啤酒糟与棉粕的配料比、料水比和发酵时间变化的二次回归方程,并利用该方程探讨了各因子对纤维素酶的影响。结果表明,各因子对纤维素酶的影响顺序为:配料比>发酵时间>料水比,而因子的交互作用不显著(P>0.05)。利用统计优选法寻优,确定了最优发酵条件:啤酒糟与棉粕的配料比7:3,料水比1:1.5,发酵时间66 h,FPA最高值为782.4 U/g。3.对该菌株产的纤维素酶的酶学性质进行研究表明:CMCase最适作用温度为60℃,最适反应pH 4.6,FPA的最适作用温度为50℃,最适反应pH为5.0。60℃热处理1 h,FPA相对酶活力为87.3%,CMCase相对酶活力为98.2%。CMCase和FPA在pH 3.0至pH 7.0之间较稳定。1 mmol/L Cu2+离子、5 mmol/L Ca~(2+)离子及1～10 mmol/L Mn~(2+)离子对酶活有极显著的激活作用(P<0.01);5 mmol/L和10 mmol/L Cu~(2+)离子对酶活具有极显著的抑制作用;Na~+、K~+、Ba~(2+)、Zn~(2+)、Mg~(2+)等金属离子在1～10 mmol/L范围内,对酶活的作用不显著(P>0.05)。
4.对发酵产物进行分析表明,发酵后干物质损失率为12.58%,粗纤维降解率为29.36%。发酵后,培养基中的鼠李糖、阿拉伯糖、木糖、甘露糖、葡萄糖、纤维二糖含量极显著提高(P<0.01)。酶系分析表明,发酵产物中具有纤维素酶、木聚糖酶、β-甘露聚糖酶、β-葡聚糖酶和酸性蛋白酶。
Brewer’s spent grain (BSG) is one of the main by-products of brewing industry. Many companies sold it as animal feed, but some companies just discharged it as waste which caused pollution to the environment and resource waste. The main components of BSG were malt hull as well as leftovers of malt after saccharification. These substances were rich in cellulose that is inducer of cellulase. In addition, BSG contained nitrogenous compounds, inorganic elements and vitamins. So it especially suits to cellulase production by solid state fermentation. Using brewer’s spent grain as the main material, we carried out cellulase production by Aspergillus niger sp. in solid state fermentation. The fermentation conditions were studied and optimized in this paper as well as the characteristics of cellulase and the effects of fermentation on medium composition were also studied. The main results are as follows:
1. The fermentation conditions were obtained through the single factor experiment.The results showed that the optimal condition was as follows: 20g brewer’s grain and cotton cake (the ratio of them was 8:2) in 500mL flask as culture medium, the ratio of material to water was 1:1.5, incubated for 66 h at 30℃. Under this condition, the maximal filter paper activity (FPA) and carboxymethyl cellulase activity (CMCase) reached 759.9±51.7 and 14187.8±579.1 U/g (dry matter), respectively. While the influence of different forms of inorganic nitrogen (ammonium sulfate, ammonium chloride, ammonium nitrate, diammonium phosphate, and urea) used in the experiment on the cellulase activity was not significant when the nitrogen content was equal, and the influence on cellulase activities was not significant after adding 0～2.5% KH_2PO_4 or 0~1.6% CaCl_2 into the medium, respectively.
2. Based on single variable experiments, cellulase production which was carried out by Aspergillus niger sp in solid state fermentation using brewer’s spent grain was optimized by quadratic general rotary unitized design. A quadratic regression equation was established, and the effects of the factors on cellulase were also studied by the equation. The results showed that the order of the three factors that affected the cellulase fermentation was the ratio of brewer’s grain to cotton cake>fermentation time>the ratio of material to water. And the mutual effect of different factors was not significant (P>0.05) . The statistical optimization was used to optimize the fermentation conditions, and the optimum condition was as follows: the ratio of brewer’s grain to cotton cake was 7:3, the ratio of material to water was 1:1.5, the time of fermentation was 66 h, the maximal filter paper activity was obtained by 782.4 U/g (dry matter).
3. The study on the enzymatic characteristics of the crude enzymes from Aspergillus niger sp. was carried out. The results show that the optimal reaction temperature of CMCase and FPA activity were 60℃and 50℃respectively. The optimal reaction pH value were 4.6 and 5.0, respectively. The FPA and CMCase kept 87.3% and 98.2% after thermal treatment for one hour when the temperature is 60℃, respectively. At the same time, the FPA and CMCase were stable from pH 3.0 to 7.0. One mmol/L Cu~(2+), 5 mmol/L Ca~(2+) and 1 to 10 mmol/L Mn~(2+) could stimulate the enzyme activities very significantly(P<0.01). Five mmol/L and ten mmol/L Cu~(2+) inhibited the enzyme activities very significantly. Na~+、K~+、Ba~(2+)、Zn~(2+)、Mg~(2+) showed no significant effect on the enzyme activities in a range of 1 to 10 mmol/L(P>0.05)。
4. The analysis of fermented product indicated that dry matter loss ratio was 12.58% and 29.36% of coarse cellulose was decomposed. The content of rhamnose, arabinose, xylose, mannose, glucose and cellobiose in the medium were increased very markedly after fermentation (P<0.01). The studies on enzyme system of Aspergillus niger sp showed that xylanase, mannaseβ-glucanase and acid protease were inclouded in the fermentated products.
1.通过单因素实验对黑曲霉发酵啤酒糟生产纤维素酶的发酵工艺条件进行了初步研究,适宜的培养基组分为:500 mL三角瓶中装入啤酒糟和棉粕20 g,配料比8:2,料水比1:1.5,于30℃发酵66 h,滤纸酶活(FPA)和羧甲基纤维素酶活(CMCase)分别达到759.9±51.7 U/g和14187.8±579.1 U/g(干物质);在含氮量相等的条件下,试验所用的(NH4)2SO4、NH4Cl、NaNO3、(NH2)2CO、(NH4)2HPO4对酶活影响不显著;KH2PO4和CaCl2分别在0～2.5%和0～1.6%的添加范围内对产酶影响也不显著。
2.在单因素研究的基础上,采用三因素二次通用旋转组合试验,研究了啤酒糟与棉粕的配料比、料水比和发酵时间对黑曲霉固态发酵啤酒糟生产纤维素酶的影响,建立了纤维素酶随啤酒糟与棉粕的配料比、料水比和发酵时间变化的二次回归方程,并利用该方程探讨了各因子对纤维素酶的影响。结果表明,各因子对纤维素酶的影响顺序为:配料比>发酵时间>料水比,而因子的交互作用不显著(P>0.05)。利用统计优选法寻优,确定了最优发酵条件:啤酒糟与棉粕的配料比7:3,料水比1:1.5,发酵时间66 h,FPA最高值为782.4 U/g。3.对该菌株产的纤维素酶的酶学性质进行研究表明:CMCase最适作用温度为60℃,最适反应pH 4.6,FPA的最适作用温度为50℃,最适反应pH为5.0。60℃热处理1 h,FPA相对酶活力为87.3%,CMCase相对酶活力为98.2%。CMCase和FPA在pH 3.0至pH 7.0之间较稳定。1 mmol/L Cu2+离子、5 mmol/L Ca~(2+)离子及1～10 mmol/L Mn~(2+)离子对酶活有极显著的激活作用(P<0.01);5 mmol/L和10 mmol/L Cu~(2+)离子对酶活具有极显著的抑制作用;Na~+、K~+、Ba~(2+)、Zn~(2+)、Mg~(2+)等金属离子在1～10 mmol/L范围内,对酶活的作用不显著(P>0.05)。
4.对发酵产物进行分析表明,发酵后干物质损失率为12.58%,粗纤维降解率为29.36%。发酵后,培养基中的鼠李糖、阿拉伯糖、木糖、甘露糖、葡萄糖、纤维二糖含量极显著提高(P<0.01)。酶系分析表明,发酵产物中具有纤维素酶、木聚糖酶、β-甘露聚糖酶、β-葡聚糖酶和酸性蛋白酶。
Brewer’s spent grain (BSG) is one of the main by-products of brewing industry. Many companies sold it as animal feed, but some companies just discharged it as waste which caused pollution to the environment and resource waste. The main components of BSG were malt hull as well as leftovers of malt after saccharification. These substances were rich in cellulose that is inducer of cellulase. In addition, BSG contained nitrogenous compounds, inorganic elements and vitamins. So it especially suits to cellulase production by solid state fermentation. Using brewer’s spent grain as the main material, we carried out cellulase production by Aspergillus niger sp. in solid state fermentation. The fermentation conditions were studied and optimized in this paper as well as the characteristics of cellulase and the effects of fermentation on medium composition were also studied. The main results are as follows:
1. The fermentation conditions were obtained through the single factor experiment.The results showed that the optimal condition was as follows: 20g brewer’s grain and cotton cake (the ratio of them was 8:2) in 500mL flask as culture medium, the ratio of material to water was 1:1.5, incubated for 66 h at 30℃. Under this condition, the maximal filter paper activity (FPA) and carboxymethyl cellulase activity (CMCase) reached 759.9±51.7 and 14187.8±579.1 U/g (dry matter), respectively. While the influence of different forms of inorganic nitrogen (ammonium sulfate, ammonium chloride, ammonium nitrate, diammonium phosphate, and urea) used in the experiment on the cellulase activity was not significant when the nitrogen content was equal, and the influence on cellulase activities was not significant after adding 0～2.5% KH_2PO_4 or 0~1.6% CaCl_2 into the medium, respectively.
2. Based on single variable experiments, cellulase production which was carried out by Aspergillus niger sp in solid state fermentation using brewer’s spent grain was optimized by quadratic general rotary unitized design. A quadratic regression equation was established, and the effects of the factors on cellulase were also studied by the equation. The results showed that the order of the three factors that affected the cellulase fermentation was the ratio of brewer’s grain to cotton cake>fermentation time>the ratio of material to water. And the mutual effect of different factors was not significant (P>0.05) . The statistical optimization was used to optimize the fermentation conditions, and the optimum condition was as follows: the ratio of brewer’s grain to cotton cake was 7:3, the ratio of material to water was 1:1.5, the time of fermentation was 66 h, the maximal filter paper activity was obtained by 782.4 U/g (dry matter).
3. The study on the enzymatic characteristics of the crude enzymes from Aspergillus niger sp. was carried out. The results show that the optimal reaction temperature of CMCase and FPA activity were 60℃and 50℃respectively. The optimal reaction pH value were 4.6 and 5.0, respectively. The FPA and CMCase kept 87.3% and 98.2% after thermal treatment for one hour when the temperature is 60℃, respectively. At the same time, the FPA and CMCase were stable from pH 3.0 to 7.0. One mmol/L Cu~(2+), 5 mmol/L Ca~(2+) and 1 to 10 mmol/L Mn~(2+) could stimulate the enzyme activities very significantly(P<0.01). Five mmol/L and ten mmol/L Cu~(2+) inhibited the enzyme activities very significantly. Na~+、K~+、Ba~(2+)、Zn~(2+)、Mg~(2+) showed no significant effect on the enzyme activities in a range of 1 to 10 mmol/L(P>0.05)。
4. The analysis of fermented product indicated that dry matter loss ratio was 12.58% and 29.36% of coarse cellulose was decomposed. The content of rhamnose, arabinose, xylose, mannose, glucose and cellobiose in the medium were increased very markedly after fermentation (P<0.01). The studies on enzyme system of Aspergillus niger sp showed that xylanase, mannaseβ-glucanase and acid protease were inclouded in the fermentated products.
引文
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