Linoleic acid, α-linolenic acid, and monolinolenins as antibacterial substances in the heat-processed soybean fermented with Rhizopus oligosporus

Article abstract of DOI:10.1080/09168451.2020.1731299

Antibacterial activities against Staphylococcus aureus and Bacillus subtilis were found in an ethanol fraction of tempe, an Indonesian fermented soybean produced using Rhizopus oligosporus. The ethanol fraction contained free fatty acids, monoglycerides, and fatty acid ethyl esters. Among these substances, linoleic acid and α-linolenic acid exhibited antibacterial activities against S. aureus and B. subtilis, whereas 1-monolinolenin and 2-monolinolenin exhibited antibacterial activity against B. subtilis. The other free fatty acids, 1-monoolein, monolinoleins, ethyl linoleate, and ethyl linolenate did not exhibit bactericidal activities. These results revealed that R. oligosporus produced the long-chain polyunsaturated fatty acids and monolinolenins as antibacterial substances against the Gram-positive bacteria during the fungal growth and fermentation of heat-processed soybean.

Full text of DOI:10.1080/09168451.2020.1731299

Bioscience, Biotechnology, and Biochemistry
ISSN: 0916-8451 (Print) 1347-6947 (Online) Journal homepage: https://www.tandfonline.com/loi/tbbb20
Linoleic acid, α-linolenic acid, and monolinolenins
as antibacterial substances in the heat-processed
soybean fermented with Rhizopus oligosporus
Dewi Kusumah, Misaki Wakui, Mai Murakami, Xiaonan Xie, Kabuyama
Yukihito & Isamu Maeda
To cite this article: Dewi Kusumah, Misaki Wakui, Mai Murakami, Xiaonan Xie, Kabuyama
Yukihito & Isamu Maeda (2020): Linoleic acid, α-linolenic acid, and monolinolenins as antibacterial
substances in the heat-processed soybean fermented with Rhizopusꢀoligosporus, Bioscience,
Biotechnology, and Biochemistry, DOI: 10.1080/09168451.2020.1731299
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BIOSCIENCE, BIOTECHNOLOGY, AND BIOCHEMISTRY
https://doi.org/10.1080/09168451.2020.1731299
Linoleic acid, α-linolenic acid, and monolinolenins as antibacterial substances
in the heat-processed soybean fermented with Rhizopus oligosporus
a
b
b
a,c
a,b
a,b
Dewi Kusumah , Misaki Wakui , Mai Murakami , Xiaonan Xie , Kabuyama Yukihito and Isamu Maeda
a
Department of Applied Life Science, United Graduate School of Agricultural Science, Tokyo University of Agriculture and Technology,
b
c
Tokyo, Japan; Department of Applied Biological Chemistry, School of Agriculture, Utsunomiya University, Utsunomiya, Japan; Center for
Bioscience Research and Education, Utsunomiya University, Utsunomiya, Japan
ABSTRACT
ARTICLE HISTORY
Antibacterial activities against Staphylococcus aureus and Bacillus subtilis were found in an
ethanol fraction of tempe, an Indonesian fermented soybean produced using Rhizopus oligos-
porus. The ethanol fraction contained free fatty acids, monoglycerides, and fatty acid ethyl
esters. Among these substances, linoleic acid and α-linolenic acid exhibited antibacterial
activities against S. aureus and B. subtilis, whereas 1-monolinolenin and 2-monolinolenin
exhibited antibacterial activity against B. subtilis. The other free fatty acids, 1-monoolein,
monolinoleins, ethyl linoleate, and ethyl linolenate did not exhibit bactericidal activities.
These results revealed that R. oligosporus produced the long-chain polyunsaturated fatty
acids and monolinolenins as antibacterial substances against the Gram-positive bacteria during
the fungal growth and fermentation of heat-processed soybean.
Received 26 December 2019
Accepted 13 February 2020
KEYWORDS
Tempe; linoleic acid; α-
linolenic acid; monolinolenin
Tempe is a heat-processed fermented soybean food,
which includes many beneficial substances for human
health [1]. The fungal fermentation process causes
many changes in nutrition, texture, and taste of soy-
bean. Many enzymes were secreted during the fermen-
tation process to break down macromolecules into
smaller molecules that have higher bioavailability.
Preceded researches have reported the nutrition,
anti-hypertension, anti-diabetic, antioxidant, antidiar-
rheal, and antibacterial contents found in tempe [2–5].
Additionally, the tempe-making process can remove
antinutrients such as antitrypsin, phytic acid, and oli-
gosaccharides from soybean [5]. The tempe extracts
protect human and animal intestinal cells from enter-
otoxigenic Escherichia coli infection [6,7].
tempe produced using R. oligosporus inhibits the
growth of S. aureus and the antibacterial activity is
dependent on the fungal growth in heat-processed
soybean [11]. Therefore, it should be hypothesized
that antibacterial substances included in the ethanol
fraction of tempe have the proteinaceous nature as
reported in the water extract from tempe produced
using R. microsporus and R. oligosporus [8,10].
In this study, purification and characterization of
antibacterial substances included in the ethanol frac-
tion of tempe produced using R. oligosporus were
performed in order to clarify their chemical structures
and bactericidal activities. A mechanism of their pro-
duction during fermentation and availability as food
preservatives in tempe were discussed.
From a food preservability viewpoint, antibacterial
activities of tempe extract have been investigated. The
water extract from tempe produced using Rhizopus
microsporus exhibits antibacterial activity against
Bacillus cereus [8]. Nullification of the bactericidal
effect in the extract after heat or protease treatment
indicates the proteinaceous nature of antibacterial
substances included in the extract. In addition, the
fractions extracted with ethanol or ethyl acetate from
tempe produced using Rhizopus oligosporus inhibit
the growth of Bacillus subtilis and Staphylococcus
aureus [9]. The tempe starter R. oligosporus can pro-
duce the 5.5-kDa peptide in submerged cultivation
broth, which inhibits the growth of Gram-positive
bacteria including B. subtilis natto, S. aureus, and
Streptococcus cremoris [10]. The ethanol fraction of
Materials and methods
Microorganisms and reagents used
The spore suspension of R. oligosporus (a starter of
ragi tempe, Raprima, Bandung, Indonesia) and
tempe were prepared as described previously [11].
S. aureus NCTC50581 (National Collection of Type
Cultures, London, UK) was cultured in Bacto tryp-
tic soy broth (Becton, Dickinson and Company,
Sparks, MD). B. subtilis ATCC6633 (American
Type Culture Collection, Manassas, VA, USA) was
cultured at 37ºC in Luria-Bertani medium (10 g/L
tryptone, 5 g/L yeast extract, 10 g/L NaCl, pH 7.2).
Oleic acid, linoleic acid, and α-linolenic acid were
CONTACT Isamu Maeda
i-maeda@cc.utsunomiya-u.ac.jp
Supplemental data for this article can be accessed here.
©
2020 Japan Society for Bioscience, Biotechnology, and Agrochemistry

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.

ANTIBACTERIAL SUBSTANCES IN TEMPE
3
Results and discussion
Identification of substances in the ethanol fraction
after fermentation
Biochemical features of antibacterial substance in
the ethanol fraction
Substances in the LC-UV peak fractions were methyl-
esterified and analyzed by GC-MS. Two major peaks
emerged around 12.7 min in the chromatograms for
peak 1, 2, 5, and 7 fractions and 12.1 min in the
chromatograms for peak 3, 4, 6, and 8 fractions, respec-
tively (Figure 2). Mass spectra of the peaks emerged at
The ethanol fraction from tempe repressed growths
of B. subtilis and S. aureus, whereas their growths
were not affected by an equal amount of ethanol.
The growth-repressing activities still remained in the
ethanol fraction after the overnight trypsin treat-
ment or boiling in water for 60 min (Supplemental
Figure. 1). A flow-through fraction in ultrafiltration
inhibited the bacterial growths (Supplemental
Figure 2), indicating that a molecular weight of
antibacterial substance might be lower than its mole-
cular weight cutoff (5 kDa). The peptide with mole-
cular weight of 5.5 kDa [10] was not observed in
sodium dodecyl sulfate-polyacrylamide gel electro-
phoresis of the ethanol fraction (Supplemental
Figure 3). These results suggest that an antibacterial
substance contained in the ethanol fraction is not
the peptide with 5.5 kDa molecular size reported in
R. oligosporus.
12.7 and 12.1 min corresponded to those of methyl
linolenate and methyl linoleate, whose molecular ions
were 292 and 294 m/z, respectively. A mass spectrum
of the peak at 12.7 min contained ions of m/z = 236
and m/z = 108 as observed in the mass spectrum of
methyl α-linolenate [14], while it did not contain ions
of m/z = 194 and m/z = 150, which were specific ions
for the mass spectrum of methyl γ-linolenate [15]. The
mass spectrum of the peak at 12.7 min was identified as
that of methyl α-linolenate. Therefore, it is suggested
that α-linolenic acyl group and linoleic acyl group are
included in the structures of substances in peak 1, 2, 5,
and 7 fractions and peak 3, 4, 6, and 8 fractions,
respectively. The ethanol fraction of tempe was ana-
lyzed by GC-MS without the esterification process.
Peaks for ethyl esters of palmitic acid, stearic acid,
oleic acid, linoleic acid, and α-linolenic acid, together
with a small peak for methyl linoleate, emerged in
a chromatograph of the fraction collected from soybean
after fermentation (Figure 3). As the fatty acid compo-
sition in soybean oil [16] corresponds to the fatty acyl
composition of the ethyl esters, these esters in ethanol
fraction might originate from fatty acids bound in
triglycerides of soybean oil. Then, LC-UV peak frac-
tions were analyzed with GC-MS without the esterifi-
cation process. Chromatograms of peak 7 and peak 8
fractions included major peaks at 12.9 and 12.3 min,
respectively, whose mass spectra corresponded to those
of ethyl linolenate and ethyl linoleate (Figure 3). The
result indicates that peak 7 and peak 8 fractions contain
ethyl linolenate and ethyl linoleate, respectively.
Analysis of substances in the ethanol fractions
The ethanol fractions collected from heat-treated soy-
beans before and after fermentation were analyzed by
the reverse phase LC. Major peaks numbered from 1
to 8 were observed in a chromatogram of the fraction
collected from soybean after fermentation, whereas
only small peaks in the retention time from peak 1 to
peak 8 were observed in a chromatogram of the frac-
tion collected from soybean before fermentation
(Figure 1). It has been shown that the antibacterial
activity against S. aureus increased significantly after
fermentation for 48 and 72 h [11]. Therefore, it is
possible that the antibacterial activities against
B. subtilis and S. aureus originate from the substances
eluted at the retention times of these peaks.
Figure 2. GC-MS chromatograms of LC-UV peak fractions after
methyl esterification.
The peak numbers of the fraction are shown at the right side. Arrows
indicate the positions where main methyl esters were eluted.
Figure 1. LC-UV chromatograms of the ethanol fractions.
Elution profiles of extracts from soybean grains before fermentation and after
fermentation are shown in broken (right scale) and solid (left scale) lines,
respectively.

4
D. KUSUMAH ET AL.
the synthesized monolinolenins (Figure 5). A molecule-
related ion was also detected as an acetic acid adduct of
deprotonated monolinolenin. From the retention times
of synthesized and commercially available compounds,
main substances included in the LC-UV and LC-MS
peaks were identified as follows: peak 1, 2-monolinolenin;
peak 2, 1-monolinolenin; peak 3, 2-monolinolein; peak 4,
1-monolinolein; peak 5, α-linolenic acid; peak a, 2-mono-
olein; peak b, 1-monoolein; peak 6, linoleic acid; peak c,
palmitic acid; peak d, oleic acid; peak e, stearic acid. It was
difficult in LC-UV to detect the substances included in
peaks a to e because of low extinction coefficients at
210 nm in an acyl group with non- or single-double
bond within their structures.
Figure 3. GC-MS chromatograms of the ethanol fractions and
LC-UV peak fractions without methyl esterification.
The peak numbers of the fraction are shown at the right side.
Minimum bactericidal concentrations of
antibacterial substances
An LC-MS chromatogram included peaks corre-
sponding to the LC-UV peaks 1 to 6 (Figure 4).
However, the peaks 7 and 8 were not observed probably
because hydrophobicity of the ester bond might make
ethyl linolenate and ethyl linoleate difficult to form their
molecule-related ions. In addition, peaks a to e were
observed in the LC-MS chromatogram. As peaks 3–4
and peaks a-b exhibited the same mass spectra as those
of commercially available 1-monolinolein and 1-mono-
olein, respectively (Figure 5). Molecule-related ions were
detected as acetic acid adducts of deprotonated mono-
linolein and monoolein. Then, monolinoleins were
synthesized from linoleic acid and glycerol. In
a chromatogram of the synthesized monolinoleins,
a peak of 1-monolinolein was higher than that of
Minimum bactericidal concentrations of fatty acids,
monoglycerides, and ethyl esters detected in the
ethanol fraction were evaluated. Linoleic acid
(peak 5) and α-linolenic acid (peak 6) exhibited
bactericidal activities against S. aureus and
B. subtilis while palmitic acid (peak c), oleic acid
(peak d), and stearic acid (peak e) did not exhibit
the activities at 1,000 µg/mL (Table 1).
Monolinolenins (peaks 1–2) exhibited an antibiotic
activity against B. subtilis, while no activities were
found in monolinoleins (peaks 3–4), monooleins
(peaks a–b), ethyl linolenate (peak 7), and ethyl
linoleate (peak 8). These results indicate that for-
mation of an ester bond in linoleic acid and α-
linolenic acid weakens their bactericidal activities.
The difference in the antibiotic activities between
monoglycerides and ethyl esters may be related to
the difference in hydrophilicity around the carbonyl
group. The mechanisms of antibacterial fatty acids
in bactericidal activities are well documented [17].
It might correlate to the amphipathic property of
fatty acids and the prime target on bacteria is the
cell membrane. At low concentrations, antibacterial
fatty acids cause pores in the cell membrane
whereas at high concentrations, they solubilize cell
membrane so that cell metabolites leak out and
cells lyse. Medium- and long-chain unsaturated
free fatty acids tend to be more active against
Gram-positive bacteria than Gram-negatives [17].
The previous studies have demonstrated the biolo-
gical effects of linoleic acid and α-linolenic acid on
inhibiting the growth of various Gram-positive bac-
teria [18,19].
2-monolinolein, because esterification in two hydroxyl
groups of glycerol results in the synthesis of 1-monolino-
lein while esterification in one hydroxyl group of glycerol
results in the synthesis of 2-monolinolein. Monooleins
and monolinolenins were also synthesized from oleic acid
or α-linolenic acid and glycerol, respectively. The synthe-
sized monooleins, monolinoleins, and monolinolenins
were purified by LC-UV while defining peaks with the lar-
ger area as peaks of 1-monoglycerides and peaks with
the smaller area as peaks of 2-monoglycerides. The LC-
MS peaks 1–2 exhibited the same mass spectra as those of
Contribution of linoleic acid and α-linolenic acid to
the antibacterial activities in tempe
During tempe fermentation, R. oligosporus produces
Figure 4. LC-MS chromatogram of the ethanol fraction from
soybean grains after fermentation.
variety of enzymes such as carbohydrase, lipase,

ANTIBACTERIAL SUBSTANCES IN TEMPE
5
Figure 5. Mass spectra of the LC-MS peaks in the chromatogram of ethanol fraction from soybean grains after fermentation.
Table 1. Evaluation of bactericidal activities of fatty acids, monoglycerides, and ethyl esters and concentrations of linoleic acid, α-
linolenic acid, and monolinolenins in tempe.
Fatty acid
1-Monoglyceride
2-Monoglyceride
Ethyl ester
18:2 18:3
1
6:0
18:0
18:1
18:2
18:3
18:1
18:2
18:3
18:1
18:2
18:3
MIC (S. aureus) (µg/mL)
MIC (B. subtilis) (µg/mL)
Concentra-tion (mg/g wet wt.)
–
–
ND
–
–
ND
–
–
ND
400
20
15.2
600
8
4.35
–
–
ND
–
–
ND
–
400
0.40
–
–
ND
–
–
ND
–
800
0.76
–
–
ND
–
–
ND
–
No bactericidal activity was found at 1,000 µg/mL.
ND, not determined.
protease, and phytase [20]. Lipase is a kind of lipolytic
enzymes that cleave fatty acid from lipid head groups
by hydrolysis [17]. In this study, the composition of
fatty acids and acyl groups in ethyl esters corre-
sponded to the fatty acid composition in soybean oil
[21]. For the food industries, the spoilage of products
with spore-forming bacilli is the main concern in
extending their shelf-life. The concentrations of lino-
leic acid and α-linolenic acid were higher than those of
monolinolenins, and three orders of magnitude higher
than their minimum bactericidal concentrations
against B. subtilis (Table 1). Furthermore, monolino-
lenins were included in tempe at the concentration
around their minimum bactericidal concentrations.
The result suggests that linoleic acid and α-linolenic
acid might mainly contribute to the intensive antibac-
terial activity especially to B. subtilis in tempe. This
[
16]. Therefore, it is likely that linoleic acid, α-
linolenic acid, and monolinolenins might be con-
verted from triglycerides in soybean oil by
R. oligosporus lipase during fermentation. Bacillus
spp. are involved in deteriorating of large varieties of
food products such as milk, dairy products, meat
products, bakery products, and fermented soybeans

6
D. KUSUMAH ET AL.
study has first demonstrated that the antibacterial-free
fatty acids and monoglycerides, whose concentrations
are especially effective against B. subtilis, are contained
in tempe produced using R. oligosporus.
[9] Mambang D, Suryanto D. Antibacterial activity of
tempe extracts on Bacillus subtilis and Staphylococcus
aureus. Jurnal Teknologi dan Industri Pangan. 2014;25
(1):115–118.
[
[
[
10] Kobayasi S, Okazaki N, Koseki T. Purification and
characterization of an antibiotic substance produced
from Rhizopus oligosporus IFO 8631. Biosci
Biotechnol Biochem. 1992 Jan;56(1):94–98. PubMed
PMID: 1368137; eng.
11] Kusumah D, Kabuyama Y, Maeda I. Promotion of
fungal growth, antibacterial and antioxidative activ-
ities in tempe produced with soybeans thermally trea-
ted using steam pressure. Food Sci Technol Res.
Author Contribution
DK and IM were responsible for the organization of research
and design of experiments. DK, MW, MM, XX, and IM were
responsible for the identification of compounds. DK, MW, MM,
KY, and IM were responsible for the syntheses of compounds
and their antibiotic activity measurements. All authors contrib-
uted to the writing of the final manuscript.
2018;24(3):395–402.
12] Seto Y, Kang J, Ming L, et al. Genetic replacement
of tesB with PTE1 affects chain-length proportions
of 3-hydroxyalkanoic acids produced through
beta-oxidation of oleic acid in Escherichia coli.
Disclosure statement
J
Biosci Bioeng. 2010 Oct;110(4):392–396.
No potential conflict of interest was reported by the authors.
PubMed PMID: 20547355; eng.
[
13] Bossaert WD, De Vos DE, Van Rhijn WM, et al.
Mesoporous sulfonic acids as selective heterogeneous
catalysts for the synthesis of monoglycerides. J Catal.
References
1999;182(1):156–164.
[
[
[
[
14] Hallgren B, Ryhage R, Stenhagen E. The mass spectra
of methyl oleate, methyl linoleate, and methyl
linolenate. Acta Chem Scand. 1959;13(4):845–847.
15] Holman RT, Rahm JJ. Analysis and characterization
of polyunsaturated fatty acids. Prog Chem Fats Other
Lipids. 1971;9:13–90.
16] Graef G, Fehr W, Miller L, et al. Inheritance of fatty
acid composition in a soybean mutant with low lino-
lenic acid. Crop Sci. 1988;28(1):55–58.
[
1] Astuti M, Meliala A, Dalais FS, et al. Tempe,
a nutritious and healthy food from Indonesia. Asia
Pac J Clin Nutr. 2000 Dec;9(4):322–325. PubMed
PMID: 24394511; eng.
[
[
2] Esaki H, Onozaki H, Kawakishi S, et al. New antiox-
idant isolated from tempeh. J Agric Food Chem. 1996
Jan 01;44(3):696–700.
3] Gyoergy P, Murata K, Ikehata H. Antioxidants isolated
from fermented soybeans (tempeh). Nature. 1964 Aug
17] Desbois AP, Smith VJ. Antibacterial free fatty acids:
activities, mechanisms of action and biotechnological
potential. Appl Microbiol Biotechnol. 2010 Feb;85
22;203:870–872. PubMed PMID: 14204076; eng.
[
[
4] Karyadi D, Lukito W. Beneficial effects of tempeh in disease
prevention and treatment. Nutr Rev. 1996;54(11):S94–S98.
5] Nout MJR, Kiers JL. Tempe fermentation, innovation
and functionality: update into the third millenium.
J Appl Microbiol. 2005;98(4):789–805.
(6):1629–1642. PubMed PMID: 19956944; eng.
[
[
18] Feldlaufer M,F, Knox D,A, Lusby W,R, et al.
Antimicrobial activity of fatty acids against Bacillus
larvae, the causative agent of American foulbrood
disease. Apidologie. 1993;24(2):95–99.
19] Kabara JJ, Swieczkowski DM, Conley AJ, et al.
Fatty acids and derivatives as antimicrobial agents.
Antimicrob Agents Chemother. 1972 Jul;2(1):23–28.
PubMed PMID: 4670656; PubMed Central PMCID:
PMCPMC444260. eng.
[
[
6] Roubos-van den Hil PJ, Nout MJ, Beumer RR, et al.
Fermented soya bean (tempe) extracts reduce adhe-
sion of enterotoxigenic Escherichia coli to intestinal
epithelial cells. J Appl Microbiol. 2009 Mar;106
(
3):1013–1021. PubMed PMID: 19191956; eng.
7] Roubos-van den Hil PJ, Schols HA, Nout MJ, et al.
First characterization of bioactive components in soy-
bean tempe that protect human and animal intestinal
cells against enterotoxigenic Escherichia coli (ETEC)
infection. J Agric Food Chem. 2010 Jul 14;58
[
[
20] de Reu JC, Ramdaras D, Rombouts FM, et al. Changes
in soya bean lipids during tempe fermentation. Food
Chem. 1994 01;50(2):171–175. Jan.
21] Abdou AM, Higashiguchi S, Aboueleinin AM, et al.
Antimicrobial peptides derived from hen egg lyso-
zyme with inhibitory effect against Bacillus species.
Food Control. 2007;18(2):173–178.
(
13):7649–7656. PubMed PMID: 20550210; eng.
[
8] Roubos-van PJ, Dalmas E, Nout MR, et al. Soya bean
tempe extracts show antibacterial activity against
Bacillus cereus cells and spores. J Appl Microbiol.
2010;109(1):137–145.