| Beef meaty peptide (BMP) is one of the main component of beef flavor, which has a good synergistic taste with sodium glutamate, can enhance the beer flavor, and has good thermal stability, suitable for the requirements of heat treatment in food industrial production. This work studied the BMP expression condition in shake-flask fermentation level and investigated the fermentation control in a5L fermentor. Different purity of BMP were obtained using several protein separation and purification methods. The function of BMP in flavor formed in Maillard reaction were investigated using BMP concentrated solution and sensory evaluation were preformed.In the shake-flask fermentation, firstly, this study investigated the effect of gene dosages on BMP expression and the target protein expression levels of4copies,8copies,12copies and16copies were10.6mg/L,16.3mg/L,22.9mg/L and35.4mg/L respectively. On this basis, this study studied the strategies of methanol additon in the methanol induction phase, the result shown that target BMP expression will be higher than before by a gradient increase addition of methanol in the1.0%,1.2%,1.4%and1.6%manner every day.In the research of expansion fermentation with fermentor. a three-step fermentation strategy were used for BMP expression, which include the glycerol culture phase, glycerol fed-batch culture phase and the methanol fed-batch culture phase. Through the optimization of glycerol fed-batch cycle, results show that maintenance the glycerol fed-batch speed of15mL/L·h for4hours could achieve the appropriate cell density and keep a good physical condition. which is conducive to the expression of exogenous proteins. Since the methanol fed-batch control based on dissolved oxygen, using restrictive fed methanol strategy by adjusting the feeding cycle and the pulse which can make the rate of methanol added equal to the rate of consumption, and the dissolved oxygen were controlled at10%25%. Under fermentation control, foreign protein expression increased55.04%(up to645mg/L) and induction time reduced24h.In the study of BMP separation and purification, using ammonium sulfate salt-out and hollow fiber membrane separation method for rough separation, respectively. Results show that using hollow fiber can interception the protein molecular weight between6kDa60kDa, the total protein recovery reached60%, while the ammonium sulfate salt-out can not make the most of the protein precipitation. Then use anion exchange resin FPA53and DEAE52, respectively, to separation concentrated BMP liquid. Results showed that the use of DEAE52can help get high-purity target protein, with an average purity as high as93.19%, while FPA53cannot. In the Maillard reaction, hollow fiber concentrated BMP solution was used as a react material while beef soup or water were used as a control, respectively. The optimization of flavor reaction formula was studied by single factor experiment with sensory evaluation as the main index. The optimazed formula is as follow:concentrate BMP liquid50mL, water50mL, xylose10g, beef fat3.0g, Cysteine1.5g, Taurine2.3g, Thiamine1.0g, mixed amino acids0.7g, NaCl2.0g. After mixing the samples, adjusted pH5.0and heated at100℃for120min, then cooled for sensory evaluation. The results showed that using concentrated BMP solution as a react material, Maillard reaction products have prominent aroma of beef, roast beef flavor, mellow taste, strong meat texture, and with significantly beef soup color. |
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