Identification of diacetonamine from soybean curd residue as a sporulation-inducing factor toward Bacillus spp.

Article abstract of DOI:10.1186/s13568-017-0395-0

Under bioassay-guided investigation, a sporulation-inducing factor (SIF) toward Bacillus spp. was searched for in methanol (MeOH) extracts of soybean curd residues, and diacetonamine (1) was identified as the active compound. SIF was first isolated as a monoacetylated derivative (2, 4.1?mg from 655?g soybean curd residues), and its chemical structure was elucidated by field desorption mass spectrometry, electron ionization mass spectrometry, and nuclear magnetic resonance (NMR) analyses. After 48-h incubation, 40?μM diacetonamine hydrochloride (1b) exhibited sporulation-inducing activity with 35% sporulation frequency toward a Bacillus amyloliquefaciens wild-type strain (AHU 2170), whereas 40?μM diacetone acrylamide (3) showed 99% sporulation induction, which was much higher than that of 1b. Although Bacillus megaterium NBRC 15308 was sporulated by the treatment with 400?μM 1b with 36 and 70% sporulation frequency after 72- and 96-h incubation respectively, 3 at the same concentration showed only 2% sporulation after 72-h incubation. Hence, diacetonamine (1) was characterized as a genuine SIF from soybean curd residues, but it was uncertain whether 1 is a natural product or an artifact. Spores of B. amyloliquefaciens induced by 1b survived after treatment with heating at 95?°C for 10?min, also suggesting that 1 is genuine SIF in soybean curd residue. As sporulation induction is likely linked to activation of antibiotic production in some spore-forming Firmicutes bacteria, compound 1 would be a possible chemical tool to develop an effective fermentation technology in Bacillus species.

Full text of DOI:10.1186/s13568-017-0395-0

et al. AMB Expr (2017) 7:101
Ikeda
DOI 10.1186/s13568-017-0395-0
Open Access
ORIGINAL ARTICLE
Identifcation of diacetonamine
from soybean curd residue as a
sporulation-inducing factor toward Bacillus spp.
Aki Ikeda1, Dongyeop Kim1,2 and Yasuyuki Hashidoko1*
Abstract
Under bioassay-guided investigation, a sporulation-inducing factor (SIF) toward Bacillus spp. was searched for in
methanol (MeOH) extracts of soybean curd residues, and diacetonamine (1) was identifed as the active compound.
SIF was frst isolated as a monoacetylated derivative (2, 4.1 mg from 655 g soybean curd residues), and its chemical
structure was elucidated by feld desorption mass spectrometry, electron ionization mass spectrometry, and nuclear
magnetic resonance (NMR) analyses. After 48-h incubation, 40 µM diacetonamine hydrochloride (1b) exhibited
sporulation-inducing activity with 35% sporulation frequency toward a Bacillus amyloliquefaciens wild-type strain
(AHU 2170), whereas 40 µM diacetone acrylamide (3) showed 99% sporulation induction, which was much higher
than that of 1b. Although Bacillus megaterium NBRC 15308 was sporulated by the treatment with 400 µM 1b with 36
and 70% sporulation frequency after 72- and 96-h incubation respectively, 3 at the same concentration showed only
2% sporulation after 72-h incubation. Hence, diacetonamine (1) was characterized as a genuine SIF from soybean
curd residues, but it was uncertain whether 1 is a natural product or an artifact. Spores of B. amyloliquefaciens induced
by 1b survived after treatment with heating at 95 °C for 10 min, also suggesting that 1 is genuine SIF in soybean curd
residue. As sporulation induction is likely linked to activation of antibiotic production in some spore-forming Firmi-
cutes bacteria, compound 1 would be a possible chemical tool to develop an efective fermentation technology in
Bacillus species.
Keywords: Sporulation-inducing factor, Soybean curd residue, Bacillus amyloliquefaciens, Bacillus megaterium,
Diacetonamine, Diacetone acrylamide
active production of BT-toxin in B. thuringiensis (de
Maagd et al. 2001; Deng et al. 2014) and some lipopep-
tide antibiotics in B. subtilis antibiotic production (Stein
2005), for example, are highly linked to their sporulation
events. Not only severe starvation stresses but also some
other physical and physiological stimulants can lead to
sporulation induction (de Vries et al. 2004; Rashad et al.
2013), and this mechanism appears similar to bioflm
formation for Gram-negative bacteria as a starvation-
tolerant state, of which morpho-diferentiation and meta-
bolic changes are regulated by auto-regulatory chemical
signals known as quormones or autoinducers (Davies
et al. 1998; Duanis-Assaf et al. 2015). Soybean curd resi-
due, also known as soybean pulp or okara, is an excellent
culture medium ingredient for antibiotic production in
Introduction
Few genera of the phylum Firmicutes, such as Bacillus,
Paenibacillus, and Clostridium, form endospores under
nutrient-starvation conditions, an event known as sporu-
lation (Higgins and Dworkin 2012). Bacterial spores are
highly tolerant to heat or drought, and even survive after
autoclaving (e.g. 120 °C for 15 min). Many functional
genes for sporulation in Bacillus spp. have been investi-
gated (Eijlander et al. 2014). Tese spore-forming Gram-
positive bacteria show not only morphological but also
metabolic diferentiation. In metabolic diferentiation,
*Correspondence: yasu-h@abs.agr.hokudai.ac.jp
1 Research Faculty of Agriculture, Hokkaido University, Kita 9 Nishi 9,
Kita-Ku, Sapporo 060-8589, Japan
Full list of author information is available at the end of the article
© The Author(s) 2017. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License
(http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium,
provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license,
and indicate if changes were made.

et al. AMB Expr (2017) 7:101
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several antibiotic-producing microorganisms (Hayashi curd residue using B. amyloliquefaciens AHU 2170 and B.
et al. 1989; O’Toole 1999; Mizumoto et al. 2006). Particu- megaterium NBRC 15308 as the test bacilli.
larly, Bacillus subtilis produces a large amount of iturins,
cyclic lipopeptide antibiotics, in soybean curd residue Materials and methods
medium (Ohno et al. 1995, 1996). Conversely, numer- General
ous reports also suggested a tight link between active Electron ionization mass spectrometry (EI-MS), feld
sporulation induction and antibiotic production among desorption mass spectrometry (FD-MS), and feld des-
Bacillus species cultured in soybean curd residues (Stein orption high-resolution mass spectrometry (FD-HR-MS)
2005).
were conducted using MS spectrometers JMS-T100GCV
Indeed, methanolic extract from soybean curd residue and JMS-SX-102 (JEOL, Tokyo, Japan). 1H-/13C-NMR
induced sporulation during pellicle formation (Fig. 1). spectra were recorded using a JEOL JNM-EX270 (JEOL,
Hence, we screened for sporulation inducing factor Tokyo, Japan) at 270/67.5 MHz and a Bruker AMX-500
(SIF) in soybean curd residues using a wild-type Bacil- (Bruker, Billerica, MA, USA) at 500/125 MHz.
lus amyloliquefaciens AHU 2170 originally isolated from
Diacetonamine hydrogen oxalate (1a) was purchased
the rhizoplane of a home garden-grown Chinese cabbage from Sigma-Aldrich (St. Louis, MO, USA), and diaceton-
and stocked in our laboratory (Sugita 2008). Its major amine hydrochloride (1b) and N-acetyldiacetonamine (2)
antifungal substances were iturin A and fengycin B (Mal- were prepared from 1a. As another chemical derivatiza-
fanova et al. 2012; Yamamoto et al. 2015), but this strain ble from 1, diacetone acrylamide (3) was purchased from
was less active in producing these lipopeptide antibiotics. Wako (Osaka, Japan), while a β-amino acid, 3-amino-
When the extracts of soybean curd residue were added to 3-metylbutyric acid (4), used as a negative control was
24-h pre-cultured nutrient-broth medium (correspond- purchased from Combi-Blocks (San Diego, CA, USA).
ing to 1 g wet soybean curd residue per mL medium), Teir chemical structures are shown in Fig. 2.
antibiotic production in the liquid medium was clearly
improved in the culture (Additional fle 1: Figure S1). Bacterial strains and culture conditions
More importantly, sporulation of B. amyloliquefaciens For the sporulation assay, we used B. amyloliquefaciens
was also accelerated, after 48-h exposure to the crude AHU 2170 isolated from the rhizosphere of Chinese
extract (Fig. 1). Given the tight linkage between antibiotic cabbage (Brassica rapa var. pekinensis) and stocked in
production and sporulation induction based on molecu- our laboratory (Sugita 2008) and B. megaterium NBRC
lar biology analyses (Eijlander et al. 2014; Deng et al. 15308 purchased from NITE Biological Resource Center,
2014), we frst searched for SIF in the extracts of soybean Kisarazu, Japan. Both strains of bacilli were pre-cultured
Fig. 1 Sporulation induction on vegetative cells of B. amyloliquefaciens AHU 2170 by an extract from soybean curd residue. An active fraction
obtained by preliminary cation-exchange chromatography induced pellicle along with sporulation induction at a concentration equivalent to 1 g
fresh weight of soybean curd residues per mL MSG medium after 48-h incubation (left), while control without any additives did not (right). Bacterial
cells were stained with SYTO 9/propidium iodide for bacterial LIVE/DEAD assay. Note that shapes of the spore cells stained by SYRO 9 are clearly
distinguishable from rod-shaped vegetative cells

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Fig. 2 Structures of diacetonamine and its related chemicals
at 30 °C for 24 h on a nutrient-broth (NB) agar plate MSG medium previously autoclaved. Te 10-mL cell sus-
before use. Te B. amyloliquefaciens wild-type strain pension was linear-shaken in a 30-mL Erlenmeyer fask
easily formed spore-based pellicle in NB liquid medium, at 80 rpm at 30 °C for 48 h for B. amyloliquefaciens. B.
whereas B. megaterium was a rarely sporulating Bacillus megaterium required 72–120 h for sporulation.
species (Strnadová et al. 1991; Larsen et al. 2014).
A LIVE/DEAD BacLight Bacterial Viability Kit
(L13152) (Lafamme et al. 2004) was used to evaluate bac-
Bioassay for sporulation
terial sporulation induction. Te bacterial cells exposed
After shaking culture of B. amyloliquefaciens or B. mega- to a test substance or fraction for the incubation time
terium in NB medium (40 mL) at 30 °C for 48 h in the were collected by brief centrifugation, and the resulting
dark as reciprocation of 100 rpm, the samples were cell pellet was washed with 1 mL saline twice by pipet-
divided into four portions (10 mL each in 15-mL Fal- ting and then suspended in 1 mL or 100 µL Milli-Q water.
con tube) and centrifuged at 3500g at 4 °C for 15 min. Te cell suspension was then mixed with 10 µL of LIVE/
Te resulting bacterial cell pellet was re-suspended in DEAD BacLight Bacterial Viability Kit solution (SYTO 9:
10 mL MSG (minimal salts mixture +0.5% glutamic acid) propidium iodide, 9:1) and incubated for 15 min at room
medium (Freese et al. 1979) containing a test substance temperature in the dark, and then centrifuged at 10,000g
equivalent to that originally contained in 10 g soybean for 1 min to remove the supernatant. Te resulting cell
curd residue, or maximum concentration at 400 µM. pellet was re-suspended in 100 or 20 µL Milli-Q water,
When 400 µM of a chemical solution should be pre- and a 3-µL portion of the resulting cell suspension was
pared, a 400 mM solution of the compound dissolved dropped on a glass slide (S1111, Matsunami Glass, Kishi-
in dimethyl sulfoxide (DMSO) was frst passed through wada, Japan) and pressed tightly with a cover glass (Mat-
a sterile Millex®-GV membrane flter (0.22 µm polyvi- sunami Glass). Te prepared slide was observed using a
nylidene fuoride, Merck Millipore Ltd., Billerica, MA), fuorescent microscope BioRevo (Keyens, Osaka, Japan)
and a 10 µL portion of the solution was added to 10 mL under 1500× magnifcation with an oil immersion lens.

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Column chromatography in Sephadex G‑25 and Sephadex
LH‑20
Spores of B. amyloliquefaciens are barrel-like short-
rods (physical size of approximately 0.8 µm × 1.2 µm)
distinguishable from vegetative cells (typical rods,
approximately 0.7 × 2.5 µm). Using a GFP-BP flter (Ex
470/40, Em 535/50, dichroic mirror 495), live cells and
spores were stained as green with SYTO 9, whereas pro-
pidium iodide with a TRITC flter (Ex 540/25, Em 605/70,
dichroic mirror 565) stained dead cells red. Competent
cells (Yang et al. 2015) were observed as orange-colored
rods. Te spore cells stained with SYTO 9 were observed
as green along the border with the unstained central part.
Sporulation frequency (numbers of spore/numbers of
spore plus vegetative cells) was calculated.
A half volume of the aqueous layer concentrated to
50 mL (over 25 g) was subjected to gel fltration using a
1.5 L Sephadex G-25 (200 g, GE Healthcare Little Chal-
font, UK) column (400 mL per fraction, more than 8 frac-
tions). Sporulation-inducing activities were observed
in the frst three fractions, from Fr-G-1 to Fr-G-3. Te
remaining half portions of the extracts were also sub-
jected to gel fltration at the same scale as the Sephadex
G-25 column, and all the active fractions obtained were
combined (Fr-G-1–3, 21.76 g).
Fraction Fr-G-1–3 was further fractionated by col-
umn chromatography using a 450-mL Sephadex LH-20
(Amersham Pharmacia Biotech AB, Uppsala, Sweden)
column by eluting with 5% MeOH (300 mL per fraction,
total of 12 fractions), and Fr-LH-2 and LH-3 (Fr-LH-2–3,
16.55 g) showed active sporulation induction toward B.
amyloliquefaciens.
Vegetative cells of B. megaterium are relatively large
(1.0 × 15 µm or more), while sporulated cells were more
spherical endospores (approximately 0.8 µm × 1.0 µm)
than those of B. amlyloliquefaciens and easily distinguish-
able from vegetative cells. Immature spores of B. megate-
rium were stained by SYTO 9, while mature spores were
not.
Cation‑ and anion‑exchange chromatography
For cation-exchange chromatography, Fr-LH-2–3 con-
taining active components were applied to a 600 mL-
bed volume of a CM-cellulose (cation exchange carrier,
Wako) open column, previously conditioned with 0.2 M
aq. HCl followed by washing with Milli-Q water. Te
active component was eluted with Milli-Q water in the
second fraction (600 mL, Fr-CMC-2) obtained as 4.08 g
of syrup.
Heat‑tolerance assay for evaluating sporulation frequency
A
100-µL portion of spore suspension diluted
101–105-fold with Milli-Q water in a polymerase chain
reaction (PCR) tube was heated at 95 °C for 10 min in
a PCR thermocycler. After cooling to room tempera-
ture, the suspension was well mixed by pipetting and
spread over a NB agar plate using a disposable bacte-
ria spreader. After 24-h incubation at 30 °C in the dark,
colonies were counted. As a blank, all bacterial cell sus-
pensions were spread on NB agar plates without heating
treatment.
Anion-exchange column chromatography using DEAE-
cellulose (Wako, 600 mL-bed volume) was subsequently
conducted for Fr-CMC-2. Te DEAE-cellulose column,
previously conditioned with 0.2 M aq. NaOH solution
followed by washing with Milli-Q water, retained the
active component in the column, despite elution with
Milli-Q water (1200 mL). Terefore, the column was con-
tinuously eluted with 600 mL of 0.2 M Na2CO3 solution.
As the concentrated solutes (Fr-DEAE-2, 9.71 g contain-
ing excessive Na2CO3 salts) exhibited sporulation induc-
tion, all solutes were subjected to a second gel fltration
step in a 400-mL Sephadex G-25 column for elution
with Milli-Q water (100 mL for each fraction). Te third
fraction (Fr-DS-3, 1.02 g) showed sporulation-inducing
activity.
Extraction of sporulation‑inducing factor (SIF)
from soybean curd residue
A 10-kg fresh weight soybean curd residue (1.85 kg
from Miyoshiya Tofu, Bibai, Japan, 2.45 kg, Mameta,
Sapporo, Japan, 3.0 kg, Marukawa Foods, Sapporo,
Japan, and 2.7 kg, At the Bran Made of Koyama, Sap-
poro, Japan) was collected from grocery shops and
mixed for extraction with methanol (MeOH). Te soy-
bean curd residues were soaked in 10 L of MeOH, and
the methanolic extract was collected by decantation and
squeezing the remaining residues in a cotton cloth. Te
squeezed residues were re-extracted with another 6 L
of MeOH. Te methanolic extracts further collected
and combined were fltered in vacuo and concentrated
under a low pressure to 500 mL of an aqueous solu-
tion. Tis aqueous solution was partitioned with hexane
(500 mL × 3, 0.7 g), ethyl acetate (EtOAc) (500 mL × 3,
2.0 g), and normal butanol (n-BuOH) (500 mL × 3,
9.6 g). Te resulting aqueous layer was the only fraction
active as SIF.
Normal phase silica gel TLC
Fraction Fr-DS-3 was analyzed by normal phase silica
gel thin-layer chromatography (TLC) (Merck Kiesel-
gel 60F254 Art. 1.05715, Merck, Kenilworth, NJ, USA)
developed in n-BuOH:water (H2O):acetic acid (AcOH)
4:1:1. Te main substance in the fraction gave a dark
brown-colored spot at Rf 0.4 by spraying with vanillin-
sulfuric acid reagent. Te main compound was obtained

et al. AMB Expr (2017) 7:101
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by preparative TLC (10 × 10 cm) in n-BuOH:H2O:AcOH of colorless syrup. EI-MS (m/z, %): 157 ([M]+, 11), 142
4:1:1 for a small portion of Fr-DS-3. However, the main ([M–CH3]+, 9.4), 114 ([M–COCH2 + H]+, 18), 100 ([M–
compound obtained and eluted from silica gel with 50% CH3-COCH2 + H]+, 94), 98 (42), 83 (57), 72 (24), 60 (22),
MeOH showed no sporulation induction. Te chemical 58 (100), and 43 (85). 1H-NMR (270 MHz, δH, in CDCl3):
substance recovered from the plate lower zones of the 5.8 (1H, br., NH), 2.94 (2H, s, CH2), 2.12 (3H, s, CH3),
main band also eluted with 50% MeOH and showed clear 1.90 (3H, s, Ac), and 1.39 (6H, s, 2 × CH3). 13C-NMR
sporulation activity.
and distorsionless enhancement by polarization transfer
Tis fraction exhibited a characteristic unique pink- (DEPT) (67 MHz, δH, in CDCl3): δC 208.18 (CO), 170.11
ish color by spraying with p-anisaldehyde-sulfuric acid (COCH3), 52.15 (C), 50.93 (CH2), 31.77 (CH3), 27.52
reagent followed by heating, but this compound showed (CH3 × 2), and 24.36 (COCH3).
a severe tailing in the acidic solvent. Tus, we replaced
AcOH with 25% aq. NH4OH solution, and the sub- Preparation of few diacetonamine derivatives
stance gave a fne spot on normal phase silica gel TLC in from diacetonamine hydrogen oxalate
n-BuOH:25% aq. NH4OH:acetone 4:5:2.
Commercially available diacetonamine hydrogen oxalate
(1a, Sigma-Aldrich) was subjected to an anion exchange
reaction. Compound 1a (1.23 g, 6 mmol) dissolved in
10 mL Milli-Q water was passed through a conditioned
Silica gel column chromatography with n‑BuOH:25% aq.
NH4OH:acetone
Te active component in Fr-DS-3 (1.00 g) was further DEAE cellulose column (200 mL bed volume). An alka-
purifed by silica gel column chromatography (174 g, line fraction eluted frst was collected and neutralized
Wakogel C60, Wako) and eluted with n-BuOH: 25% using 1 M aq. HCl solution and then concentrated to
aq. NH4OH: acetone (4:5:2). Silica gel column was frst yield 0.82 g of diacetonamine hydrochloride (1b) as a
conditioned with Milli-Q water followed by the eluting colorless syrup (90% yield).
solution, and then Fr-DS-3 was loaded to obtain frac-
For the acetylation reaction, 1a (196.4 mg) dissolved in a
tions (25 mL each). Each fraction was immediately con- 1:1 mixture of Ac2O and pyridine (4 mL) was incubated at
centrated in vacuo. Te target substance responsive to 70 °C for 1 h. A major product, N-acetyldiacetonamine (2),
p-anisaldehyde-sulfuric acid reagent was detected in was obtained by preparative TLC (134.2 mg, 89% yield) as
Fr-15–Fr-22 as a single spot on TLC. All fractions (SIF- a pinkish purple-colored spot upon spraying with p-anisal-
Fr-15-22) together (450 mg of a colorless solid) exhibited dehyde-sulfuric acid spray reagent followed by heating.
signifcant sporulation-inducing activity at a concen-
tration equivalent to 1 g soybean curd residue per mL Assay for diacetonamine and its amide derivatives
medium (45 µg SIF-Fr-15-22 per mL).
Diacetonamine hydrochloride (1b) was preliminarily
FD-MS (m/z, %) of SIF-Fr-15–22 showed several par- tested at concentrations of 4, 40, and 400 µM for spor-
ent ion peaks, such as 407.0 (42), 385.0 (20), 308.1 (21), ulation-inducing activities toward B. amyloliquefaciens
215.0 (25), 193.0 (100), and 116.1 (81). We also tested sev- and B. megaterium. All the test compounds, including
eral coloring reagents for the detection of active spots on 1b, N-acetyldiacetonamine (2), and diacetone acrylamide
TLC, including p-anisaldehyde-sulfuric acid reagent and (3), were dissolved in DMSO to be 400 mM, and diluted
ninhydrin. Te target compound showed a pale pink color with MSG medium into fnal concentrations of 4, 40,
on spraying with p-anisaldehyde-sulfuric acid reagent fol- and 400 µM. Te MSG medium for the sporulation assay
lowed by strong heating, but no response to ninhydrin.
always contained 0.1% DMSO.
Acetylation of SIF‑fraction
Results
Although the active component was highly concentrated Isolation of SIF and its chemical behaviors
in SIF-Fr-15–22, the active fraction contained a relatively Te SIF toward B. amyloliquefaciens was chased in the
high amount of silica gel and contaminants. To remove extract of soybean curd residue (Scheme 1). Using the
inactive contaminants, a portion of the dried active frac- sporulation-inducing assay, sporulation-inducing activ-
tion was subjected to an acetylation reaction. A portion ity was detected in the water-soluble layer, as those
of SIF-Fr-15–22 (29.5 mg) re-dissolved in 2 mL of acetic equivalent to 10 g soybean curd residue in 10 mL MGS
anhydride and pyridine (1:1) was incubated at 70 °C for medium. Gel fltration of the water layer (more than 50 g)
1 h. One of the main products, 2, was detected at an Rf in Sephadex G-25 column resulted in early elution of the
0.15 on silica gel TLC (hexane:EtOAc 1:1) as a purple- active substance (Fr-G-1–3, total of 21.76 g). Subsequent
colored spot by spraying with p-anisaldehyde-sulfuric chromatography for Fr-G-1–3 in Sephadex LH-20 col-
acid reagent. Tis product was purifed by preparative umn further fractionated active substance in early frac-
TLC developed twice in hexane:EtOAc 1:1 to yield 4.0 mg tions (Fr-LH-2–3, 16.55 g).

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Scheme 1 Overall steps for isolation of sporulation-inducing factor from soybean curd residue
We attempted cation- and anion-exchange column chro- fraction yielding the target compound as a single spot on
matography for Fr-LH-2–3. By elution with Milli-Q water, TLC (n-BuOH:25% aq. NH4OH:acetone 4:5:2) showed
the active substance was completely passed through the cat- clear sporulation induction activity. Approximate calcu-
ion exchange CM-cellulose column, but elution of the active lation by the bioassay suggested that at least 1/10 of the
component was clearly retarded from the void fraction (Fr- active substance was recovered from the extracts of 10 kg
CMC-2, 4.08 g). Te active fraction Fr-CMC-2 was further soybean curd residue.
applied to the anion exchange DEAE-cellulose column.
By elution with Milli-Q water, the active substance was Identifcation of diacetonamine as active SIF from soybean
captured in the column and eventually eluted with 0.2 M curd residue
Na2CO3 solution. After removing Na2CO3 salt by a second FD-MS analysis of SIF-Fr-15–22 showed several par-
gel fltration step in a Sephadex G-25 column, the active ent ion peaks, indicating that this active fraction was a
fraction (Fr-DS-3, 1.02 g) gave one major spot on normal mixture. A portion of the active component (29.5 mg)
phase silica gel TLC (n-BuOH:H2O:AcOH 4:1:1). However, was subjected to acetylation. After this reaction, SFI-
this acidic and oligomeric sugar-like spot was inactive.
Fr-15–22 yielded two major products, and the one
In contrast, a weak purple lower spot on TLC, appear- detected at Rf 0.15 in hexane:EtOAc 1:1 showed a pinkish
ing on spraying with p-anisaldehyde-sulfuric acid, was color by spraying with p-anisaldehyde-sulfuric acid rea-
active in the sporulation assay. We further focused on gent. Tis target product (4.1 mg of colorless syrup, 30%
this substance obtained as a fne condensed purple spot yield) showed its parent ion at m/z 157 (115 + COCH2),
on TLC in n-BuOH:25% aq. NH4OH:acetone 4:5:2. All indicating a monoacetylated product derived from com-
Fr-DS-3 was subjected to silica gel column chromatogra- pound with a molar mass of 116.1 (chemical formula of
phy by eluting with the same solvent as used for TLC. Te C6H13NO + H+, by FD-HR-MS).
fractions (Fr-15–22) containing the target substance were
1H-, 13C-NMR, and DEPT spectra of the monoacety-
combined (SIF-Fr-15–22, 450 mg of colorless solid). Tis lated derivative (1c) in chloroform-d showed two singlet

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methyl proton signals at δH 1.39 magnetically equiva- obtained by the treatment with 40 µM of 3 were sub-
lent (CH3 × 2, s), singlet methyl proton signal at δH 1.90 jected to heat-tolerant assay, and almost all of the spores
assignable as the acetyl group (–NH–CO–CH3), another survived at 95 °C (Fig. 4). Tus, appearance of the B.
singlet methyl proton signal at δH 2.12 assignable as amyloliquefaciens colonies showed that the spores thus
connected with the carbonyl carbon on the skeleton (– induced by 3 and 1c are genuine endospores resistant to
CO–CH3), and set of methylene proton signals equiva- highly adverse conditions.
lent at δH 2.94 (s, 2H).
In the vegetative cells of B. megaterium treated with
13C-NMR and DEPT showed two equivalent methyl 400 µM of 1b, sporulation induction (36%) was observed
carbons at δC 27.52 (×2), methyl carbon next to the at 72-h incubation, in which spores were uniquely stained
carbonyl group (δC 31.76), and methylene carbon at δC with SYTO 9 as green fuorescence (arrowheads in 72 h,
50.93. A quaternary carbon at δC 52.15 was assigned as Fig. 5a). After the incubation for 96–120 h, the spores of B.
connected with an amino group and two methyl groups. megaterium have changed to mature one unstainable with
Carbon signals at δC 170.11 (carboxy carbon) and δC SYTO 9 (arrowheads in 120 h, Fig. 5a). Tus, relatively
24.36 (methyl carbon) were assignable to an acetyl group. slow response of B. megaterium to 1b needed 72-h or
Tus, one remaining carbon for the chemical formula longer incubation for the sporulation induction. In addi-
(C6H13NO + COCH2) was assigned as a carbonyl carbon tion, dose-dependent manner of 1b revealed that 40 µM
(δC 208.18) positioned between the methyl carbon (δC of 1b induced relatively early stages of sporulation toward
31.77) and methylene carbon (δC 50.93).
vegetative cells of B. megaterium after 96-h incubation
Te monoacetylated compound, derivatized from (55%), while 400 µM of 1b induced 70% sporulation to B.
the active principle, was thus elucidated as N-acetyl-4- megaterium under the same incubation conditions. Con-
amino-4-methyl-2-pentanone (= N-acetyldiacetonamine, versely, 1b at 4 µM did not induce any sporulation at 96 h
2). Terefore, the compound was identifed as diaceton- or even after the incubation for 120 h (Fig. 5b).
amine (4-amino-4-methyl-2-pentanone, 1) with a molar
Diacetone acrylamide (3) was also examined its spor-
mass of 115.1 g/mol. From commercially available diace- ulation inducing activity toward B. megaterium. Com-
tonamine hydrogen oxalate (1a), N-acetyldiacetonamine pound 3 at 400 µM did not induce any sporulation at
(2) was also prepared (yield 89% from 1a) and completely 72-h incubation (Fig. 6a). By the incubation for additional
1
matched with 2 prepared from SFI-Fr-15–22 in the H- 24 h, 3 led to 66% sporulation frequency that was almost
and 13C-NMR spectra.
equivalent to 1b at the same concentration (400 µM)
(Fig. 6b). Tus, 3 showed slower sporulation induction
than 1b, leading to almost a 24-h delayed response of B.
megaterium in the morpho-diferentiation. Te negative
Activity of diacetonamine (1) and diacetonamine amide
derivatives
Using relatively stable diacetonamine hydrochloride (1b), control 4 at the same concentration did not induce any
the sporulation-inducing activity of 1 was examined. As sporulation even at 120 h incubation.
our isolation process for compound 1 allowed approxi-
mate estimation that 1 kg soybean curd residue contains Discussion
at most 45 mg (approximately 400 µmol) of 1. Hence, Our study revealed that diacetonamine (1) was the main
400 µM was initially set at the concentration of pure 1b active component in soybean curd residue. Isolation and
tested for sporulation induction toward the vegetative identifcation of 1 as SIF were successfully achieved due
cells of B. amyloliquefaciens. Within 48 h of incubation, to our simple and quick bioassay system using the B.
compound 1b at 400 µM showed sporulation inducing amyloliquefaciens wild-type strain. In this study, diacet-
activity (51% frequency) nearly equivalent to that of the onamine hydrochloride (1b) showed greater than 34 and
fnal active fraction from soybean curd residue, but its 51% sporulation frequencies toward vegetative cells of B.
1/10 concentration (40 µM) was less active (35% sporula- amyloliquefaciens at 40 and 400 µM respectively, while
tion frequency) (Fig. 3; Table 1).
40 µM of diacetone acrylamide (3) induced nearly 100%
As compound 1 with the free amine is relatively unsta- sporulation frequency in the same sporulation assay.
ble, two amide derivatives N-acetyldiacetonamine (2) Conversely, vegetative cells of B. megaterium upon expo-
and diacetone acrylamide (3) more stable than 1 were sure to 400 µM of 1b needed more than 72-h incubation
also tested for sporulation induction assay. Toward B. for sporulation induction. As B. megaterium is a Bacillus
amyloliquefaciens incubated for 48 h, 400 µM of 2 and 3 species difcult to induce sporulation (Larsen et al. 2014),
showed 54 and 69% sporulation frequencies respectively, the sporulation-inducing activities of 1b and 3 toward
which were almost equivalent to or obviously higher than this bacterium are a reliable evidence for 1 as the SIF
that of 1b. Interestingly, 40 µM of 3 showed 99% sporu- from soybean curd residue. In addition, the sporulation-
lation without cell death (Fig. 3). Terefore, the spores inducing activities of 1 and its related compounds toward

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Fig. 3 Spore-inducing activity of diacetonamine and its amide derivatives toward B. amyloliquefaciens AHU 2170. Sporulation after the treatment
with 40 and 400 µM is shown in microscopic photographs. Spores are apparent as short rods stainable as green by SYTO 9. Photo-images are after
48-h incubation upon exposure to the test compounds in MSG medium under a fuorescent microscope. Sporulation frequency was defned as ( ),
ambiguously positive as less than 10% sporulation from all the vegetative cells; (+), weakly positive as 10–50% sporulation; and (++), positive as
>50% sporulation. Scale bar = 5 μm
B. megaterium suggested that not only B. amyloliquefa- used for the extraction solvent (Nakamoto et al. 1976).
ciens but several Bacillus species are responsive to 1 for In contrast, it is most likely that the substance 1 isolated
sporulation induction.
from soybean curd residue is not the artifact spontane-
So far as our literature investigation, an aqueous ously produced during the isolation process of SIF, sim-
extract of Pueraria radix contained diacetonamine-rei- ply because we used neither acetone nor ammonia for the
neckate (Nakamoto et al. 1975). However, the authors extraction and following purifcation process to obtain
have also shown that 1 in the extract was an artifact Fr-DS-3 (Scheme 1). Indeed, Fr-DS-3 allowed detection
yielded by Michael addition of ammonia to mesityl of 1 as a unique purple spot on TLC by spraying with
oxide that had been formed by dimerization of acetone p-anisaldehyde-sulfuric acid reagent followed by heating.

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Table 1 Sporulation frequency of B. amyloliquefaciens and B. megaterium
Species (conditions)
B. amyloliquefaciens (%, 48 h)
B. megaterium (%, 72 h)
B. megaterium (%, 96 h)
Compound (concentration)
1b (40 µM)
1b (400 µM)
2 (400 µM)
3 (40 µM)
3 (400 µM)
4 (400 µM)
Control
35
51
54
99
69
6
–
55
70
−
85
66
−
7
36
−
−
2
−
0
1
Sporulation frequency (%) is calculated by counting spores and vegetative cells in randomly selected diferent views. (−) Not tested. More than 300 cells were counted
and classifed into spores and vegetative cells. For 1b and 3, sporulation assays were repeated trice to confrm their reproducible sporulation inducing activities
Fig. 4 Heat-tolerant assay for sporulated B. amyloliquefaciens AHU 2170 with diacetone acrylamide. A 50 µL portion of bacterial cell suspension
exposed to 40 µM diacetone acrylamide, incubated for 48-h, and 100-times diluted with Milli-Q water, was kept at 95 °C for 10 min and spread over
a 9-cm nutrient broth agar plate. Incubation at 30 °C for 48 h. Two“control”plates (left) are without any chemical treatments, while two plates of“not
heated”(bottom) are without heating treatments
Conversely, there is no evidence whether the compound
As a report on biological activity of 1 or its deriva-
1 is a spontaneous artifact during food processing for tives, 1 oxalate-copper has been characterized as an
production of soybean curd residue or a secondary antifungal substance (Ji et al. 2009). Conversely, diac-
metabolite contained in soybean endosperm. Hence, we etone acrylamide (3) that has been produced on an
never eliminate possibility that 1 is produced as an arti- industrial scale polymerizes with a wide variety of vinyl
fact during boiling and pasting processes of imbibed compounds for a crosslinking agent was characterized
soybean.
as a stable sporulation inducer. Since 3 is water-soluble

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a
b
Fig. 5 Spore-inducing activity of diacetonamine hydrochloride toward B. megaterium. a Time-course (48–120 h) of sporulation in B. megaterium
NBRC 15308 exposed to 400 µM of diacetonamine HCl (1b). Open arrowheads in photo-images at 72 and 120 h show immature (stained with SYTO
9) and mature (unstained with SYTO 9) endospores, respectively. b Dose-dependent manner of 1b (4, 40, and 400 µM) in the sporulation of B.
megaterium. At 96-h incubation, microscopic photo-images were recorded. Scale bar = 5 μm
and more stable than 1, amide derivatives including 3 event is often linked to the high productivity of antibi-
would be a promising target for a photoafnity probe otics (Stein 2005), our discovery of diacetonamine (1) as
design to detect sporulation signal-sensing proteins a SIF-like compound may contribute to rapid prepara-
linked to the sporulation (Asayama and Kobayashi 1993; tion of probiotic materials and exploiting an efective
Lecca et al. 2016). Because it is known that sporulation fermentation technology for antibiotic production by

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a
b
Fig. 6 Comparison of sporulation-inducing activities of diacetonamine hydrochloride (1b) and diacetone acrylamide (3) toward B. megaterium. For
the sporulation induction assay using B. megaterium NBRC 15308, SIF-like activities of the test compounds (1b, 3, and 4) were examined at 400 µM.
a Sporulation induction to B. megaterium vegetative cells at 72-h incubation. b Sporulation induction at 96-h incubation. Arrowheads show typical
spores abundant in each bacterial cell culture. Scale bar = 5 μm

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Additional fle 1: Figure S1. Activity of methanolic extract from soybean
curd residues in antibiotic production.
Abbreviations
AcOH: acetic acid; DMSO: dimethyl sulfoxide; EI-MS: electron ionization mass
spectrometry; EtOAc: ethyl acetate; FD-HR-MS: feld desorption high-resolu-
tion mass spectrometry; FD-MS: feld desorption mass spectrometry; MeOH:
methanol; MSG: glutamine-containing minimal salt; NB: nutrient-broth;
n-BuOH: normal butanol; NMR: nuclear magnetic resonance; SIF: sporulation-
inducing factor; TLC: thin-layer chromatography.
Authors’ contributions
AI did isolation of active substance from the methanolic extract, and also
evaluated diacetonamine and its related compounds for sporulation induc-
tion. DK collected preliminary data for chromatographic behavior and
biological activity of the SIF in soybean curd residues, and established simple
assay to evaluate sporulation induction using SYTO 9. YH initially planned
and designed experiments, and identifed SIF in the extract of soybean curd
residue as diacetonamine. YH wrote manuscript and DK assisted it. All authors
read and approved the fnal manuscript.
Lafamme C, Lavigne S, Ho J, Duchaine C (2004) Assessment of bacterial
endospore viability with fuorescent dyes. J Appl Microbiol 96(4):684–692.
doi:10.1111/j.1365-2672.2004.02184.x
Author details
1 Research Faculty of Agriculture, Hokkaido University, Kita 9 Nishi 9, Kita-Ku,
Sapporo 060-8589, Japan. 2 Present Address: School of Dental Medicine, Uni-
versity of Pennsylvania, 240 South 40th Street, Philadelphia, PA 19104, USA.
Larsen N, Thorsen L, Kpikpi EN, Stuer-Lauridsen B, Cantor MD, Nielsen B,
Brockmann E, Derkx PM, Jespersen L (2014) Characterization of Bacillus
spp. strains for use as probiotic additives in pig feed. Appl Microbiol
Biotechnol 98(3):1105–1118. doi:10.1007/s00253-013-5343-6
Lecca P, Mura I, Re A, Barker GC, Ihekwaba AEC (2016) Time series analysis of
the Bacillus subtilis sporulation network reveals low dimensional chaotic
dynamics. Front Microbiol 7:1760. doi:10.3389/fmicb.2016.01760
Malfanova N, Franzil L, Lugtenberg B, Chebotar V, Ongena M (2012) Cyclic lipo-
peptide profle of the plant-benefcial endophytic bacterium Bacillus sub-
tilis HC8. Arch Microbiol 194(11):893–899. doi:10.1007/s00203-012-0823-0
Mizumoto S, Hirai M, Shoda M (2006) Production of lipopeptide antibiotic
iturin A using soybean curd residue cultivated with Bacillus subtilis in
solid-state fermentation. Appl Microbiol Biotechnol 72(5):869–875.
doi:10.1007/s00253-006-0389-3
Nakamoto H, Miyamura S, Inada K, Nakamura N (1975) The study of the aque-
ous extract of Puerariae radix. I. The preparation and the components of
the active extract. Yakugaku Zasshi 95(2):1123–1127. https://www.jstage.
jst.go.jp/article/yakushi1947/95/9/95_9_1123/_pdf
Nakamoto H, Inada K, Nakamura N (1976) The study on the aqueous extract
of Puerariae radix. III. On the formation of diacetonamine from choline-
reineckate in acetone. Yakugaku Zasshi 96(2):251–254
Acknowledgements
We thank Mr. Yusuke Takada (GC–MS and NMR Laboratory, Research faculty of
Agriculture, Hokkaido University) for MS and NMR analyses. We would like to
thank Editage (www.editage.jp) for English language editing.
Competing interests
The authors declare that they have no competing interests.
Availability of data and materials
Available.
Ethics approval and consent to participate
This article does not contain any studies with human participants or animals
performed by any of the authors.
Funding
This work was supported by JSPS KAKENHI Grant Number JP 26252058.
Ohno A, Ano T, Shoda M (1995) Production of a lipopeptide antibiotic, surfac-
tin, by recombinant Bacillus subtilis in solid state fermentation. Biotechnol
Bioeng 47(2):209–214. doi:10.1002/bit.260470212
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in pub-
lished maps and institutional afliations.
Ohno A, Ano T, Shoda M (1996) Use of soybean curd residue, okara, for the
solid state substrate in the production of a lipopeptide antibiotic, iturin
A, by Bacillus subtilis NB22. Process Biochem 31(8):801–806. doi:10.1016/
S0032-9592(96)00034-9
Received: 20 April 2017 Accepted: 26 April 2017
O’Toole DK (1999) Characteristics and use of okara, the soybean residue
from soy milk production—a review. J Agric Food Chem 47(2):363–371.
doi:10.1021/jf980754l
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