Studies On Complex Enzymes For Brewing

The complex enzyme used in beer brewing is mainly comprised by β-glucanase, proteinaseand α-amylase. β-glucan is disassembled by β-glucanase specially during the diastatic action.Viscosity and turbidimetry of the wort is decreased,the velocity of filtration was increased and aswell as the fermentable substrates, so β-glucannase plays an important role in beer brewing.In this thesis, the screening of β-glucanase high-producing strain was conducted. Thefermentation process and β-glucanase character were also investigated consequently. The mainresults are as follows:1. β-glucanase hyper-producer was derived from the parental Bacillus amyloliquefacienswihc was stored in our lab by UV and 60Co mutation methods. A new mutant strain BS-5582was achieved with β-glucanase activity was 119.2U/mL in the flask fermentation, it was increasedby 34% compared with the parent stratin after the single colony separation, plate screen and flaskfermentation experiment. The new mutant strain was stable for the β-glucanase activityproduction.2. The effects of medium composition and fermentation condition on β-glucanase productionin flask fermentation were considered. The optimal medium compositions (g/L) were determinedas: barley cake flour 20, corn flour 40, bean cake flour 40, Na2HPO4 12, NH4Cl 0.75, (NH4)2 SO44, CaCl2 1. The preferred conditions for the batch fermentation in the flask were also investigated.The initial pH of medium was 7.0, fermetation temperature was 37 ℃, pitching volum was 8.0%and fermentation time was 60h. Adding 0.1% Tween-80 could increase the β-glucanase activity.β-glucanase activity produced by the new mutant strain BS-5582 was 164.6U/mL under theoptimization condition,which was increased by 38.1% compared with before. Protenase activitywas 3210.9U/mL.3. In 5L automated jar fermentor, β-glucanase activity was increased by 600 r/min ofagitation speed. It was reached 122.7U/mL with 36h fermentation time. If the fermetation pH waskept at 7.0, it was not good for the β-glucanase activity.4. It was investigated in the 8000L fermentor sacle. The strain culture in the 1000L tank wasas follows: agitation speed 600r/min, culture temperature 37℃, tank pressure 0.08MPa;the 8000Lfermentor condition was: agitation speed 160r/min, fermentaoin time 40～42h, the activity ofβ-glucanase, α-amylase and protenase was 146.7U/mL, 76.6U/mL and 13132.8U/mL,respectively.5. It was investigated preliminarily for the β-glucanase character by the fermentation liquid.Its optimum pH was 6.5. The activity could be maintained at high level during pH 6.0-7.0 and itcould be stable during pH 5.5-6.5. The optimal action temperature was 55℃, it could bemaintained at high level during 40-60℃. Some iron such as Ca2+,Na+,NH4+,K+ and Mg2+ wasdone good for β-glucanase activity, while the Cu2+,Fe2+ was harmful for its activity. Zn2+ wasdone little influence on its activity.