Glucose (Guangdong Guanghua Technology Co., Ltd, batch number: 20200403); yeast extract (Beijing Aoboxing Bio-tech Co., Ltd, batch number: 20200422); calcium carbonate (Shanghai Titan Scientific Co., Ltd, batch number: P1260108); agar (Beijing Solarbio Technology Co., Ltd, batch number: 310C022); anhydrous alcohol (Shanghai MacLean Biochemical Technology Co., Ltd, batch number: C11974944); chromium chloride (Shanghai MacLean Biochemical Technology Co., Ltd, batch number: C10717130); zinc chloride (Shanghai MacLean Biochemical Technology Co., Ltd, batch number: C10730413); 50-mL centrifuge tubes and EP tubes (Jiangsu Lexinkang Medical Equipment Co., Ltd); streptozotocin (STZ) (Shanghai McLean Biochemical Technology Co., Ltd, batch number: C20PA038100B); citric acid (Shanghai McLean Biochemical Technology Co., Ltd, batch number: C10723907); sodium citrate (Shanghai McLean Biochemical Technology Co., Ltd, batch number: C10712912); universal pH indicator paper (Hangzhou Test Three Technology Co., Ltd); metformin hydrochloride tablets (Beijing Jingfeng Pharmaceutical Group Co., Ltd, batch number 2004032); glucose test strips (ACCU-CHEK; Roche Diabetes Care GmbH, batch number: 26020933, etc); specific pathogen-free (SPF) C57BL/6 mice, aged 6-8 wk (Changsha Tianqin Biological Co., Ltd); A. aceti, number: GIM1.67 (Guangdong Microorganism Conservation Centre); and MIN6 cells (China Centre for Type Culture Collection) were used. The Animal Experiment Ethics Approval Number was 20200620.
Detection of coenzymes and metals in A. aceti
Collection of A. aceti: Cell suspensions (100 mL) of A. aceti were removed and distributed across a sterile 96-microwell plate, with the culture solution used as a blank control. The absorption of the suspension and the A. aceti culture solution were measured at 600 nm using the microplate reader and calculated as OD1 and OD2, respectively, with the final OD value of the A. aceti suspension taken as the difference OD1-OD2. The A. aceti suspension was centrifuged at 8000 rpm for 10 min to remove the supernatant; the precipitate (the A. aceti) was then washed once with 1 mL sterile PBS and centrifuged at 13000 rpm for 1 min to remove the supernatant. Thereafter, the precipitate was weighed and resuspended with sterile water for a final concentration of A. aceti at 0.1 mg/mL.
Detection of chromium and zinc in A. aceti: Collected samples were sent to Shanghai WEIPU Chemical Technology Service Co., Ltd, and the contents of chromium and zinc were detected by inductively coupled plasma mass spectrometry.
Detection of NAD+/NADH in A. aceti: The concentrations of NAD+/NADH in the A. aceti was determined using the NAD+/NADH assay kit with WST-8 (Beyotime Biotechnology).
Detection of glucose dehydrogenase in A. aceti: Solarbio’s “glucose dehydrogenase microplate assay kit” was used to detect glucose dehydrogenase concentrations in A. aceti, according to the instruction manual.
Construction of a diabetes mouse model: For the preparation of citrate buffer, 2.10 g of citric acid was treated with 100 mL of double distilled water to make a citric acid mother liquor (solution A), after which 2.94 g of trisodium citrate was treated with 100 mL of double-distilled water to make a sodium citrate mother liquor (solution B). Solution A and solution B were mixed in a ratio of 1:1.32 (or 1:1), and the pH value was measured with a pH meter and adjusted from 4.2 to 4.5. This represented the 0.1 mol/L sodium citrate-hydrochloric acid buffer solution required to prepare streptozotocin (STZ).
Seventy SPF grade C57BL/6J female mice, aged 6 wk and weighing 20 ± 2 g, were allowed to eat and drink without restrictions during 5 d of adjustable feeding. Six mice were randomly selected as the normal control group, and the rest were used for model construction.
To prepare the STZ required to inject the mice to cause diabetes, 24 mL of 0.1 mol/L sodium citrate buffer was treated with 120 mg of STZ away from the light (equivalent to a concentration of 5 mg/mL) and placed in an ice environment. The mice were made to fast for 10 h and then treated with STZ at a dose of 0.15 mL/10 g of mouse weight (equivalent to 75 mg/kg). STZ was administered by intraperitoneal injection for 3 consecutive days. Before each intraperitoneal injection, the STZ liquid was carefully pipetted with a 1 mL syringe to mix the precipitate before it was extracted to maintain the STZ concentration. After each intraperitoneal injection, mice were deprived of food and water for 90 min. On the 7th d after the last administration (the mice were made to fast for 10 h before blood collection), blood was collected from their caudal veins, and the fasting blood glucose (FBG) levels of the mice were measured with a Roche glucometer. Pathological models of mice with diabetes were confirmed as having been successfully established when FBG ≥ 16.7 mmol/L.
The diabetic mice were designated from high to low, according to their blood glucose levels, and the model mice were divided into a model group (PBS), positive control group (metformin), metal chromium plus zinc group (concentrations of chromium and zinc calculated as 1 × 10-7 mg/mL and 2 × 10-8 mg/mL, respectively, according to the highest content of chromium- and zinc-rich A. aceti), A. aceti group (OD = 1), and chromium- and zinc-rich A. aceti group (OD = 1). There were six mice in each group, and each was given 0.5 mL of treatment by gavage.
Evaluation of hypoglycemic activity in vivo: After the diabetic models were successfully established, the mice were given intragastric administration of treatment on the second day, once a day, for 15 consecutive days. The normal control group and the model group were given the same amount of PBS-water. The positive drug control group was given diformin tablets (ground into powder, prepared into a suspension with reverse osmosis water, and administered intragastrically at 0.320 g/kg/d). The metal chromium plus zinc group was given chromium trichloride (1 × 10-7 mg/mL) and zinc chloride (2 × 10-8 mg/mL) intragastrically. Through general observation, the activity and spirit of the mice, their eating and drinking, urine and feces, and the dryness and wetness of the bedding were observed every day during the 15 d of administration. Starting at the beginning of treatment, fasting blood glucose was measured every 3 d. After 15 d of administration, the mice were fasted for 10 h and blood was collected from the eyeballs. Thereafter, the mice were sacrificed, with the tissue taken from their pancreas islets, fixed with formaldehyde, and processed for hematoxylin and eosin (HE) staining and immunohistochemistry with the apoptosis-related Bax proteins. Tissue from pancreatic islets was also prepared for electron microscopy to analyze the ultrastructural damage and possible repair on tissues and cells of pancreatic islets. The weights of mice were recorded every 3 d.
Evaluation of MIN6 cell growth: A. aceti was collected (OD = 10), sonicated, and stored at -20 °C for later use. MIN6 cells, a pancreatic islet cell line, were revived, the cell concentration adjusted to 1 × 105 CFU/mL, and then cultured in 5 mmol/L and 25 mmol/L high-glucose 1640 medium on a 96-well plate with 90 mL/well. After 12 h, the cells were diluted 10-fold and dosed with 10 mL of collected A. aceti (with or without metal enrichment; OD = 10), the dose of which was equivalent to 1 × 107 CFU/mL of bacteria. A positive drug control group (diformin tablets) and negative drug control group (PBS) were included. Approximately 24 h after dosing, cell growth was detected with the CCK-8 kit (Beyotime Biotechnology).
Determination of insulin secretion in MIN6 cells: A. aceti were collected (OD = 10), sonicated, and stored at -20 °C for later use. MIN6 pancreatic islet cells were revived, the cell concentration adjusted to 1 × 105 CFU/mL, and cultured in 5 mmol/L and 25 mmol/L high-glucose 1640 medium on a 6-well plate with 1.98 mL/well. After 12 h, the cells were dosed with 20 mL A. aceti (with or without metal enrichment; OD = 10), the dose of which was equivalent to 1 × 107 CFU/mL of bacteria. A positive drug control group (diformin tablets) and negative drug control group (PBS) were included. Some 24 h after dosing, the cell supernatant was collected, with the insulin content detected by a commercially-available enzyme-immunized mouse insulin ELISA kit.