| The detection method for fructose 1, 6-diphophate (FDP) by ion chromatograph was developed in the thesis. After that, the content of FDP in the extracts of yeast cells broken by eight different methods was determined by the way, respectively. Moreover, the optimization of drying conditions for waste beer yeasts and the optimum medium for FDP fermentation with beet molasses as carbon source were carried out. The main results were as follows:1. A new method for rapid and accurate detection of FDP present in fermentation broth was established by using ion chromatography. The qualitative and quantitative analysis of FDP in the fermentation broth would be finished in 5 min under the condition with an AS11-HC anion chromatographic column, suppressed conductivity detection, and 50 mmol/L KOH elution. The minimum detection limit of the method for FDP was 0.032μmol/L (S/N=3). A significant linear relationship existed in the range with a FDP concentration from 3.3 to 211.5μmol/L (r = 0.9999, p <0.0001).The recovery of this detecting method was at 99.0% ~ 100.3% and the detecting precision was at less than 0.04%. Also, this detecting method could simultaneously be used for qualitative analysis of PO43-, fructose-6-phosphate, and glucose-6-phosphate in the fermentation broth and had no significant difference from the enzymatic analysis method of FDP.2. FDP contents of the extracts from the waste beer yeast cells broken by eight methods, including acid schizolysis, alkaline schizolysis, strong salt schizolysis, microwave treatment, autolysis, vortex-mini-bead schizolysis, ultrasound schizolysis and supermicro breaking method, were determined using ion chromatographic method. The FDP of cell extracts from three kinds of treating methods, including microwave treatment, vortex-mini-bead schizolysis and supermicro breaking method, exhibited more in content, being 0.0021, 0.0065 and 0.0048 mmol/L, respectively. Meanwhile, the treated yeast cells by the three methods above presented better permeability and stained rates by methylene blue, being 56.3%, 95%, 100%, respectively. However, the FDP-Ca2 preapared using the cell extracts from the three methods was proved to have no FDP by ion chromatographic method. This suggested that there was little FDP in the waste beer yeasts and the production of FDP extracted directly from the waste beer yeasts was of no economic returns.3. The permeability and ability of accumulating FDP of dried yeasts from four preparing methods were evaluated, including treatment by drum dryer, by forced air, freeze-drying in vacuum condition,freeze-drying at room temperature. The best preparing method was freeze-drying in vacuum condition, due to the yeast cells having higher permeability and consuming capacity of inorganic phosphate in transformation of FDP. Then, the optimization of centrifugal conditions for obtaining the fresh yeast and freeze-drying time were carried out and the optimizating condition was centrifugation at 4℃, 6, 000 g for 30 min and freeze-drying for 24 h.4. The optimum medium for FDP fermentation with beet molasses as carbon source was developed by using the active dry yeast. The optimum medium was consisted of beet molasses 98.66 g/L, NaH2PO4 75 g/L and hot active dry yeast 100 g/L. Furthermore, the fermentation technics was optimized basing on the optimum medium. The terminal of fermentation was definited at 11 h,with a higher FDP concentration of 0.267 mmol/L in fermentation broth, also a shorter time of 1.5 h and higher FDP content of 4.71% than those under the fermentation condition of using sucrose as carbon source. |
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