2
D. KUSUMAH ET AL.
purchased from Sigma-Aldrich (Tokyo, Japan).
Palmitic acid, stearic acid, and 1-monoolein
were purchased from Tokyo Chemical Industry
esterification solution (methanol-chloroform-sulfuric
acid, 10:10:0.3 (vol/vol/vol)) was added to fatty acid
or an evaporated LC-UV fraction in a screw-capped
test tube with poly(1,1,2,2-tetrafluoroethylene) (PTFE)
liner. The test tube was heated in a hole of aluminum
block placed on a hot stirrer at 94°C, 200 rpm for
(
Tokyo, Japan). 1-Monolinolein was purchased
from AccuStandard (New Haven, CT, USA).
3
0 min. The reaction mixture was cooled down to
room temperature, and 1 mL of water containing
.9% (wt/vol) NaCl was added. The test tube was
Preparation of the ethanol fractions from
heat-processed soybean and tempe
0
Soybean grains treated with steam pressure before
or after fermentation were lyophilized and finely
ground by a pestle and mortar. The ground powder
was suspended in 2.5 mL ethanol/g dry wt. The
suspension was vigorously agitated at 4°C for 8 h
and centrifuged at 13,000 × g for 10 min. The
supernatant was used as the ethanol fraction from
soybean or tempe and stored at −20°C.
vigorously agitated with a vortex mixer and centri-
fuged at 1,700 × g for 10 min. The organic phase was
collected using a Pasteur pipette, and 1 µL of the
organic phase was injected to GC-MS (Focus GC-
DSQII, Thermo Fisher Scientific, Yokohama, Japan),
which equipped a capillary column TC-70 (0.25 mm
I.D. × 60 m, GL Science, Tokyo, Japan). Helium
(>99.99995%) was used as a carrier gas at a flow
rate of 2.0 mL/min, and thermal conditions of col-
umn oven were 75°C for 2 min, 25°C/min to 180°C,
Characterization of an antibacterial substance in
the ethanol fraction of tempe
6°C/min to 240°C, and 240°C for 2 min.
The ethanol fractions were treated with 50, 100, 250,
and 500 µg/mL trypsin, and incubated for 4 h and
overnight. Conical tubes containing the ethanol frac-
tion were placed in boiling water for 5, 30, 45, and
Synthesis of monoglycerides and ethyl esters
Monoglycerides were synthesized using p-toluenesul-
fonic acid [13]. Fatty acid and glycerol were mixed at
an equal molar ratio in a screwcap test tube with
PTFE liner, and p-toluenesulfonic acid was added
after measuring 5 wt % of the mixed glycerol. The
test tube was heated in a hole of aluminum block
placed on a hot stirrer at 112°C, 200 rpm for 5 h.
Monoglycerides were dissolved in an adequate volume
of chloroform. Ethyl esters were synthesized as
described in the method of acid-catalyzed methyl
esterification with an exception of the use of ethanol
instead of methanol. Monoglycerides or ethyl esters
dissolved in chloroform were injected multiple times
to LC-UV for purification. After collecting fractions,
the eluting solvent was evaporated, and the dried
residues were dissolved in adequate volumes of etha-
nol after weighing so that concentrations of monogly-
cerides and ethyl esters were adjusted to 100 mg/mL.
6
0 min. The ethanol fractions after trypsin or boiling
treatment were added to S. aureus and B. subtilis
cultures after measuring 1.0% (vol/vol) of the cul-
tures. Bacterial growths were monitored at absor-
bance 600 nm.
Analyses by liquid chromatography (LC)
Test samples were filtrated using HLC disk 3, 0.45 μm
pore size (Kanto Chemical, Tokyo, Japan). LC-
ultraviolet (UV) absorption was performed using an
LC system, which was composed of 1525 binary
pump, 717 plus autosampler, and 2996 photodiode
array detector (Nihon Waters, Tokyo, Japan), equipped
with InertSustain C18 column (5 μm, 4.6 × 250 mm, GL
Sciences, Tokyo, Japan). Substances in the test samples
were eluted at a flow rate of 1.0 mL/min with a gradient
of water containing acetic acid (pH 3.5) and methanol
from 50% to 100% (vol/vol) methanol for 40 min and
Determination of minimum bactericidal
concentrations
1
00% methanol from 40 to 60 min. LC-mass spectro-
metry (MS) was performed using an LC system, which
was composed of LC-20AD XR pumps, SIL-20A XR
autosampler, CTO-20AC column oven, CBM-20A
communication bus module, and LCMS-2020 mass
spectrometer (Shimadzu, Kyoto, Japan), as performed
in LC-UV. Negative ions formed through atmospheric-
pressure chemical ionization were detected.
Overnight cultures of S. aureus and B. subtilis were
inoculated at one-hundredth volume of liquid medium.
The commercially available fatty acids and 1-monoolein
and the synthesized and purified monolinoleins, mono-
linolenins, 2-monolein, and ethyl esters were used as
test compounds. The test compounds dissolved in etha-
nol were added within one-hundredth of culture
volume or less at the concentrations indicated, and the
bacterial cells were exposed to the test compounds with
Analysis by gas chromatography (GC)
150 rpm shaking at 37°C for 18 h. After the exposure to
The acid-catalyzed methyl esterification was per-
formed as described previously [12]. One milliliter
test compounds, 50 µL of cell suspension was spread on
plate medium in order to evaluate bacterial viability.
Kusumah, Dewi
