Erythritol

Erythritol (99%), xylitol (≥99%),
potassium persulfate (K₂S₂O₈, 99%),
acetonitrile (99%), and pyruvic acid (≥99%) were obtained from
Sigma-Aldrich. α-Methylglyceric acid (M311505, 98%) was purchased
from Toronto Research Chemical s (TRC), Inc. Formic acid (≥99%)
was purchased from Thermofisher Scientific. Lactic acid (90% in water)
was purchased from Acros Chemicals. All reagents were used without
further purification. Compounds were dissolved and diluted using ultrapure
H₂O from a Milli-Q purification system (Millipore Sigma,
18 MΩ cm^–1). 2-Methyltetrol (MT), 1,2-dihyroxyisoprene
(1,2-DHI), 2-methyl-1,2,3-trihydroxy-4-sulfate (MT-4-S), and 2-methyl-1,2-dihydroxy-3-sulfate
anion (MD-3-S) were synthesized and purified according to the literature;^{37 (link)} their nuclear magnetic resonance (NMR) spectra
matched previously reported data. The organosulfates were purified
for use over an ion-exchange column (Dowex 50WX4, 50–100 mesh).
2-Methyl-2,3-dihydroxy-1,4-dinitrate (MD-1,4-DN) and 2-methyl-1,2,4-trihydroxy-3-nitrate
(MT-3-N) were synthesized previously by our group.^{27 (link)} All synthesized chemicals used in this work had purities
>95%, as determined by ¹H NMR. Due to the relative rate
technique and the accurate-mass or structure-specific analytical measurements,
organic impurities are not expected to alter the reaction kinetic
ratios.

All procedures involving animals were approved by the Ethics Committee for animal studies at Kyungpook National University, Republic of Korea (Approval No. KNU 2017-93). Male C57BL/KsJ-db/db (4-wk old, n = 36) were purchased from Jackson Laboratory (Bar Harbor, ME, USA). We selected male mice as experimental animals to avoid positive metabolic control by female hormones in diabetes [45 (link)]. All mice were maintained in a room with controlled temperature (20–23 °C) and lighting (alternating 12 h periods of light and dark) and fed a pelletized commercial non-purified diet for one week after arrival. Mice were then randomly divided into three groups (n = 12) and fed experimental diets for 16 weeks (Supplementary Table S1) All mice were fed normal control diet (American Institute of Nutrition AIN-76 semisynthetic diet). The control group (ND) received diet without supplementation. Experimental groups received normal diet supplemented with either Erythritol (ER, 5% (w/w) substituted for sucrose in normal diet) or d-allulose (AL, 5% (w/w) substituted for sucrose in normal diet). Erythritol was purchased from sigma Aldrich (Saint Louis, MO, USA) and d-allulose was supplied by CJ CheilJedang Corp. (Seoul, Korea).

To determine sugar and sugar alcohol contents, the washed mycelium was dried at a constant temperature, and the fermentation broth was freeze-dried. Samples (100 mg or 1 mL) were extracted in 10 ml of ultra-pure water at 100°C for 2 h, passed through a 0.45-μm filter (Millipore, Bedford, MA, United States), and then analyzed using an IC2500 HPAEC-PAD system with a GP50 quaternary gradient pump, an LC30 column oven, an EG50 automatic eluent generator, an ED50 electrochemical detector and a Dionex CarboPac MA1 column (Dionex, Sunnyvale, CA, United States). The column temperature was 30°C, and the mobile phase was 480 mM NaOH solution at a flow rate of 0.4 mL/min. Six standard external substances were used, including arabitol, mannitol, trehalose, erythritol, galactose, and glucose (Sigma, United States). Each standard substance was dissolved in deionized water and diluted in a series of standard solutions to obtain a calibration curve (Zhou et al., 2012 (link)).