Yeast extract

All chemicals were used as received without further purification. Glutathione (reduced form, 98% purity), methotrexate (MTX, 98.0% purity), methanol (99.7% purity), D2O (99.9% purity), and silver nitrate (99.9% purity) were purchased from FUJIFILM Wako Pure Chemical Corporation Ltd. (Osaka, Japan). Sodium borohydride (NaBH4, 99.99% purity) was purchased from Sigma-Aldrich (St. Louis, MO, USA).
Bacterial strains used in this study were S. mutans ATCC 35668, A. actinomycetemcomitans ATCC 29522, and P. gingivalis ATCC 33277. These strains were kept frozen until analysis. Bacterial stocks were anaerobically and statically incubated in brain heart infusion (BHI) broth (Pearlcore®, Eiken Chemical, Co., Ltd., Tokyo, Japan) supplemented with 0.1% antibiotics (gramicidin D and bacitracin, FUJIFILM Wako Pure Chemical Corporation Ltd.) and 1% sucrose (FUJIFILM Wako Pure Chemical Corporation Ltd.) for S. mutans; 1% yeast extract (FUJIFILM Wako Pure Chemical Corporation Ltd.) for A. actinomycetemcomitans; and 0.5% yeast extract, 0.0005% hemin, and 0.0001% menadione for P. gingivalis. Anaerobic incubation was carried out in an AnaeroPack system using anaerobic jars (Mitsubishi Gas Chemical Company, Inc., Tokyo, Japan).

Colonies of microorganisms that inhibited the spread of A. fumigatus on agar plates were collected to construct a library with anti-Aspergillus activity. Briefly, a piece of an agar slant with a lawn of a test microorganism was placed on a CM-agar plate 1 cm from a drop of a solution containing 1 × 10 3 spores of A. fumigatus. CM medium contained glucose 0.5%, soluble starch (Nacalai Tesque, Kyoto, Japan) 1.5%, yeast extract (Wako Pure Chemical Industries, Osaka, Japan) 0.5%, KCl 0.02%, MgSO 4 Á 7H 2 O 0.02%, KH 2 PO 4 0.1%, NaNO 3 0.2% and Bacto agar 2%. After incubation for 1-2 weeks at 20 °C, we selected microorganisms that formed colonies that inhibited the growth of A. fumigatus. These microorganisms were cultured on a CM-agar slant (4 ml) for 7 days at 20 °C. An equal volume of acetone was added to the slant, which was incubated overnight at room temperature, to extract secondary metabolites. After centrifugation, the supernatant was concentrated under reduced pressure, and distilled water was added to adjust the volume to 400 μl.

The effects of d-allulose on bacterial growth and acid production were examined in a medium constituted from 4 mL of tryptone-yeast extract-glucose (TYG) broth, which is made up of 0.5% tryptone (Becton Dickinson), 0.5% yeast extract (Wako Pure Chemical Industries, Tokyo, Japan), 1% sodium succinate, 1% sodium chloride, and 1% glucose. The broth was adjusted to a pH of approximately 6.3. The broth was inoculated with a 1% (vol/vol) bacterial suspension of the test strain and incubated at the appropriate growth temperature for 1 to 2 d under batch conditions.
At the end of the incubation, the molar growth yield on glucose (Y G ) was calculated by dividing the dry weight of cells by the molar mass of consumed glucose, as determined below. To measure dry weight, cells were collected by centrifugation (1,800 × g, 20 min, 4°C), washed once with distilled water, dried at 105°C for 4 h, and weighed.