TECHNOLOGY FOCUS - BIOTECHNOLOGY - Preferred Process: Preferably, the process for preparing demethylated rapamycin with large Bacillus comprises: (a) preparing transforming solution of the large Bacillus transforming rapamycin: inoculating the large Bacillus rapamycin stored by freezing milk into the inclined face of the nutrient agar; culturing at 35 degrees C for 15 hours; reaching the inclined face with lawn; selecting platinum ring to inoculate into the seed culture medium; where 100 ml of culture medium contains 204 g of dissolvable starch, 0.1 g of glucose, 0.3 g of yeast extract, 0.3 g of peptide, 0.1 g of dipotassium hydrogen phosphate, and 0.05 g of magnesium sulfate; after shaking culturing at 28 degrees C with speed of 220 rotations/minute for 15 hours, inoculating into the seed culture medium with the preparation identical to the preparation above according to the inoculating amount of 2.5%; shaking culturing at 28 degrees C with speed of 220 rotations/minute for 8 hours; inoculating into the transforming culture medium with identical preparation; shaking culturing at 28 degrees C with speed of 220 rotations/minute for 15 hours; adding 15 mg/ml of rapamycin mother liquid prepared with ethanol so that the final concentration is 125 mg/ml; transforming culturing the rapamycin at identical temperature and rotary speed; stopping transforming after 64 hours; and collecting the transforming solution; (b) extracting and detecting the transforming solution: collecting the transforming solution; extracting for 2 times according to a ratio of 1:1 of the transforming solution to the ethyl acetate, 20 minutes for each time; collecting the ester phase; depressurizing concentrating at 50 degrees C to remove the ester; adding ethanol solution; spanning in whole wave length with a Shimadzu LC-2010CHT efficient liquid chromatograph; where a 0DSC18 5 mu m of chromatographic column takes a fluid phase of dissolving medium of acetonitrile, methanol, and water with ratio of 55:5:40; and high performance liquid chromatography (HPLC) detecting at 50 degrees C with rate of 1 ml/minute; where the ratio of three demethylated rapamycins in the transforming solution, 29,42-O-double-demethylated rapamycin, 42-O-demethylated rapamycin, and 29-O-demethylated rapamycin is 9:10:6:19; and (c) extracting and purifying the transformed product: extracting 5 L of transforming solution with 1.5 times of ethyl acetate for 2 times, 10 minutes each time; depressurizing concentrating the ester phase at 50 degrees C; adding 100 ml of acetone to dissolve the fat soluble constituent; filtering with a No.4 core funnel to remove the insoluble substance; depressurizing concentrating at 50 degrees C; extracting the concentrated solution with 2 times of volume of ethyl acetate; depressurizing concentrating at 50 degrees C to obtain 3 ml of rough product; chromatographing with a reverse silica gel with padding of YMC-C18, 50 mu m, diameter of 2 cm, and height of 60 cm; washing with a solution of acetonitrile and water with ratio of 50:50 to 55:45; HPLC detecting; collecting the needed constituent; depressurizing concentrating each of the constituents at 50 degrees C to remove the acetonitrile; extracting the water with 2 times of ethyl acetate for 2 times; combining the extracting solution; depressurizing concentrating at 50 degrees C; chromagraphing with reverse silica gel; obtaining 92% of 29,42-O-double-demethylated rapamycin, 94% of 42-O-demethylated rapamycin, and 91% of 29-O-demethylated rapamycin; concentrating each of the constituents; respectively adding acetone with volume ratio of acetone to n-hexane of 25:75; 300 mesh silica gel column chromatographing; gradient washing with a solution of acetone and n-hexane with ratio of 25:75 to 33:67; thin layer chromatography (TLC) and HPLC tracking detecting; collecting the needed constituent; depressurizing concentrating at 50 degrees C until it is dry; obtaining 36 mg of 96% of 29,42-O-double-demethylated rapamycin, 56 mg of 98% of 42-O-demethylated rapamycin, and 21 mg of 95% of 29-O-demethylated rapamycin.