Research Of Cellulase And Its Enzymatic Characteritics Produced Using Brewer's Spent Grain Through Solid Fermentation By Aspergillus Niger Sp. And Analysis On Fermentated Products

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% KH₂PO₄ or 0¹.6% CaCl₂ 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 Cu²⁺, 5 mmol/L Ca²⁺ and 1 to 10 mmol/L Mn²⁺ could stimulate the enzyme activities very significantly(P<0.01). Five mmol/L and ten mmol/L Cu²⁺ inhibited the enzyme activities very significantly. Na⁺,K⁺,Ba²⁺,Zn²⁺,Mg²⁺ 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.