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BACKGROUND At present, there is no ideal method to cure diabetes, and there are few reports on the treatment of diabetes with probiotics. AIM To propose a method for preparing a new type of chromium- and zinc-rich Acetobacter aceti (A. aceti) and explore its ability to enhance the hypoglycemic effects of probiotics in the treatment of diabetes. METHODS A. aceti was cultured in a liquid medium that contained chromium trichloride and zinc chloride, both at a concentration of 64 mg/mL, with the initial concentration of the bacterial solution 1 × 104 CFU/mL. After the bacterial solution had been inducted for 48 h, the culture media was changed and the induction was repeated once. The levels of chromium and zinc in the bacteria were detected by inductively coupled plasma mass spectrometry, and the contents of NADH and glucose dehydrogenase were determined using an NAD/NADH kit and glucose dehydrogenase kit, respectively. Streptozotocin was used to establish a mouse model to evaluate the hypoglycemic effects of the proposed chromium- and zinc-rich A. aceti. Ten-times the therapeutic dose was administered to evaluate its biological safety. The effect on MIN6 islet cells was also assessed in vitro. RESULTS The levels of chromium metal, metallic zinc, NADH coenzyme, and glucose dehydrogenase in A. aceti prepared by this method were 28.58-34.34 mg/kg, 5.35-7.52 mg/kg, 5.13-7.26 μM, and 446.812-567.138 U/g, respectively. The use of these bacteria resulted in a better hypoglycemic effect than metformin, promoting the repair of tissues and cells of pancreatic islets in vivo and facilitating the growth of MIN6 pancreatic islet cells and increasing insulin secretion in vitro. Ten-times the therapeutic dose of treatment was non-toxic to mice. CONCLUSION Chromium trichloride and zinc chloride can be employed to induce the preparation of chromium- and zinc-rich A. aceti, which can then promote the hypoglycemic effect found in normal A. aceti. The bacteria biotransforms the chromium and zinc in a way that could increase their safety as a treatment for diabetes.