Abstract: Beer is a popular alcoholic beverage with microbiological stability. Though hop compound, alcohol, low pH, an extremely reduced content of oxygen in the environment of beer can prevent from most of microbial growth, the contamination of microbe during the producing process of beer is inevitable. The hop compounds derived from hop plant Humulus lupulus can impart a bitter flavor to beer and protect beer against spoilage. The hop compounds interfere with plasma membrane and dissipate the transmembrane pH gradient of Lactobacillus brevis. L. brevis 49 newly isolated from the commercially finished beer was used for this study, which shared 99% homology with L. brevis ATCC 14869 and ATCC 367. In order to maximally acquire proteomic information of L. brevis 49, the extraction methods and digestion conditions were specially investigated. For intracellular proteins, four factors including disruption method, the type of protease, digestion time and the amount of enzyme were optimized by applying the orthogonal test. For extracellular proteins, the nitrogen source of culture medium and precipitation method were optimized. Consequently, ultrasonication was chosen to disrupt bacteria. The combination of trypsin and chymotrypsin with the ratio of 1∶50 (enzyme to protein) was selected for digestion of intracellular proteins. 12 h was optimal for the incubation time. Under the optimized conditions, 527 intracellular proteins of L. brevis 49 were obtained. 0.6% yeast extract powder as a single nitrogen source in MRS broth was tested to be the optimized medium to extract the extracellular protein. The best extraction method for extracellular proteins of L. brevis 49 was trichloroacetic acid (TCA) method. 44 extracellular proteins with signal peptide were obtained.