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The effect of lipids on bongkrekic (Bongkrek)

acid toxin production by Burkholderia
cocovenenans in coconut media
Rafael A. Garcia
Published online: 06 Dec 2010.

To cite this article: Rafael A. Garcia (1999) The effect of lipids on bongkrekic (Bongkrek) acid toxin
production by Burkholderia cocovenenans in coconut media, Food Additives & Contaminants, 16:2, 63-69, DOI:
10.1080/026520399284217

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 Food Additives and Contaminants, 1999, Vol. 16, No. 2, 63 69
The e ect of lipids on bongkrekic (Bongkrek) acid toxin
production by Burkholderia cocovenenans in coconut media
Rafael A. Garcia, Joseph H. Hotchkiss and Keith
H. Steinkraus
Institute of Food Science, Department of Food Science, Stocking
Hall, Cornell University, Ithaca, NY 14853, USA
(Received 2 March 1998; revised 26 June 1998; accepted 30
July 1998)
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Tempe bongkrek is an Indonesian food made by
fermentation of coconut presscake or coconut milk
residue Rhizopus oligosporus. Consumption of tempe
bongkrek is associated with a food-borne human in-
toxication and signi cant numbers of deaths annually.
The bacterium Burkholderia cocovenenans, which is
the causative organism, produces two toxins, toxo avin
and bongkrekic acid (also commonly referred to as
bongkrek acid). The reasons why these poisonings
occur only in a very limited number of foods and only
in isolated regions of the world are unclear. Our
preliminary experiments in de ned media and coconut
investigated several compositional and environmental
factors and suggested that lipid type and/or concentra-
tion were important. The e ect of lipid concentration
and fatty acid type on the production of bongkrekic
acid by B. cocovenenans was examined by adding
di erent amounts of coconut fat or individual free fatty
acids to defatted and sterilized Rich Coconut Media
(dRCM). The dRCM with added lipid was inoculated
with B. cocovenenans, incubated at 30 C for 5 days
and the amount of bongkrekic acid formed quanti ed
by HPL C. Coconut fat concentrations of 10% (dry
basis) or less did not result in detectable amounts of
bongkrekic acid even though the B. cocovenenans grew
to high levels. Forty and 50% coconut fat resulted in as
much as 1.4 mg/g bonkrekic acid (dry weight) at the
same level of growth. Of eight saturated fatty acids
tested, only lauric (12:0), myristic (14:0), and palmi-
tic (16:0) acids stimulated the production of detectable
amounts of toxin. When four 18-carbon free fatty acids
with di erent degrees of saturation were compared,
signi cant amounts of bongkrekic acid (2.62 mg/g dry
weight) were produced only with oleic acid (18:1).
These data indicate that the concentration and type of
0265-203X/99 $1200
lipid in the substrate is critical for bongkrekic acid
formation. This may explain why bongkrekic acid
intoxication is limited to certain foods. Outbreaks
associated with foods containing less than 20% fat
may be a result of toxo avin formation and not
bongkrekic acid formation.
Keywords : bongkrekic acid, toxo avin, tempe
bongkrek
Introduction
Consumption of tempe bongkrek is responsible for a
signi cant number of fatal human intoxications (Con-
con 1988). The bacterium Burkholderia cocovenenans,
which can produce two toxins, bongkrekic acid (also
commonly referred to as bongkrek acid) and toxo-
avin, causes bongkrekic intoxication. B. cocovene-
nans has been isolated from tempe bongkrek,
fermented corn our, the edible fungus Tremella
fuciformis, and soil (Hu et al. 1984, Zhao et al.
1988, 1995). The intravenous LD50 for toxo avin in
mice is 1.7 mg/kg and 8.4 mg/kg when administered
orally (Lijmbach et al. 1970). The intravenous LD50
for bongkrekic acid in mice is approximately 1.4 mg/
kg and an oral dose of 6.84 mg/kg produced death in
100% of animals tested (van Veen and Mertens 1933,
Welling et al. 1960, van Veen 1966, Lijmbach et al.
1970, Hu et al. 1984).
The onset of symtoms occurs within a few hours of
ingestion of contaminated food. The patient com-
plains of malaise, abdominal pains, dizziness, exten-
sive sweating, fatigue, and eventually, coma. Death
can occur within 20 h of the onset of symptoms (Hu et
al. 1984). Both bongkrekic acid and toxo avin may
play a role in poisonings. However, bongkrekic acid is
likely to be the more important toxin. In addition to
being more toxic, the concentration of bongkrekic
acid is typically ten times higher than toxo avin (Ko
and Kelholt 1981, Ko 1985, Buckle and Kartadarma
1990).
1999 Taylor & Francis Ltd
 64 R. A. Garcia et al.
Bongkrekic poisoning has been known for more than
100 years. Virtually all reported cases occur in In-
donesia and China. In Indonesia between 1951 and
1975 there was an average of 288 reported cases per
year that resulted in an average of 34 deaths per year.
From 1961 to 1979, China reported an average of at
least one outbreak a year (Hu et al. 1984, Ko 1985,
1986, Zhao et al. 1990). Other countries in the region,
such as Cambodia and Thailand, have not reported
outbreaks but it is unclear if they do not occur or go
unreported.
Tempe bonkrek (also known as tempe bungkil kelapa
or tempe kapuk) is made from either coconut milk
residue or coconut presscake (Steinkraus 1996).
Coconut presscake is the material remaining after
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the extraction of oil from dehydrated coconut by
pressing while coconut milk residue is the material
remaining after extraction of coconut milk from fresh
coconut meat with water (Onwudike 1996). To pro-
duce tempe bongkrek, the soaked coconut presscake
or coconut milk residue is steamed for 30 60 min,
cooled, and innoculated with Rhizopus sp. It is
divided into small portions, wrapped in banana
leaves, and fermented for 48 h at ambient tempera-
ture. The nal product is a solid cake that has white
mycelial growth throughout. The cakes average 25
50 g, and several may be eaten at a single meal (van
Veen 1966, Shurtle and Aoyagi 1977, Steinkraus
1996). Most tempe bongkrek is made in homes by
individuals rather than commercially.
Tempe bongkrek made from coconut milk residue
appears to be responsible for most food poisoning
outbreaks. Coconut milk residue and presscake have
been mixed in a variety of proportions and the
mixtures inoculated with both Rhizopus oligosporus
and B. cocovenenans. Only cakes with > 20% coco-
nut milk residue produced tempe cakes that re-
sembled contaminated tempe bongkrek (cakes were
not analysed for toxins). This may be due to the
di erence in lipid content of cocunut presscake and
coconut milk residue, the latter having considerably
more residual fat (van Veen 1966, Shurtle and
Aoyagi 1977).
Corn our has been implicated in food poisoning
outbreaks in parts of China where people produce
sour fermented our. The corn is soaked for 2 4
weeks in water at room temperature with no inten-
tional inoculum. The corn is ground into our and
used to produce dumplings, noodles and bread. B.
cocovenenans contamination has been implicated but
neither bongkrekic acid nor toxo avin have been
identi ed in contaminated food so it is not known
which, if either, toxin is responsible (Zhao et al. 1990,
1995).
Tremella fuciformis (white jelly leaf, shikokikurage,
snow fungus, silver ear or white wood ear) is a fungus
that is cultivated in Japan, China, southern Taiwan
and other Asian countries and has been implicated in
bongkrekic intoxications. One study found that ap-
proximately 50% of cultivated Tremella fuciformis
was contaminated with B. cocovenenans (Tu 1981,
Hu et al. 1984, Zhao et al. 1990, Hood 1992). As
with corn, whether bongkrekic acid, toxo avin, or
both are responsible has not been determined.
B. cocovenenans may grow without the concurrent
production of bongkrekic acid. Bongkrekic acid pro-
duction is in uenced by several factors in addition to
growth of the microorganism. It is produced opti-
mally at 22 30 C and strongly inhibited at 37 C even
though this is the optimum growth temperature (Ko
1985). Production is high, in the pH range 6.5 8.0,
and drops o rapidly outside this range (Ko 1985).
An NaCl concentration of 2% inhibits bongkrekic
acid production but not growth (Buckle and Karta-
darma 1990). On coconut-based media, bongkrekic
acid production is highest 24 48 h after inoculation.
Maximal toxin levels have been reported at 2 3 or 3 6
days after inoculation (Ko 1985, Buckle and Karta-
darma 1990).
The e ect of growth media composition on bong-
krekic acid production is not well understood. Di er-
ences in substrate could help explain why outbreaks
are con ned to a very limited number of foods and
regions. Many fermented foods appear to be at risk
for toxin production. Ontjom and tempe kedele,
which are made from fermented peanut presscake
and soybeans and related to tempe bongkrek, have
not been implicated in bongkrekic acid poisonings.
Soybean tempe does not support bongkrekic acid
production (Ko 1985).
The e ects of temperature, pH, moisture and salt
concentration on bongkrekic acid production have
been investigated. However, these factors fail to ex-
plain why certain foods regularly become toxic and
other, similar foods do not. The e ect of lipid com-
position and concentration seem particularly interest-
ing given the di erences in incidence between coconut
presscake and coconut milk residue. Our objective
was to explore the link between lipid composition and
concentration on bongkrekic acid production by B.
cocovenenans in coconut media. Speci cally, the
 E ect of lipids on bongkrekic acid toxin production
e ects of fat and fatty acid concentration, fatty acid
chain length, and degree of fatty acid saturation were
investigated.
Methods
Bacterial culture
B. cocovenenans (ATCC 33664, American Type
Culture Collection, Rockville, Maryland), that was
originally isolated from contaminated tempe bong-
krek was used in all experiments (van Damme et al.
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1966). Gram stain, oxidase test, and microscope ex-
amination were used to con rm identity (Collins et al.
1989). Cultures of the organism were stored at 80 C
in glycerol until needed.
Bonkrekic acid standard
Professor J. A. Duine (Delft University of Tech-
nology, The Netherlands) generously donated a
bongkrekic acid standard. The identity of the com-
pound was con rmed by comparing its UV spectrum
(Beckman model DU640 Spectrophotometer) to the
published spectrum (Ko and Kelholt 1981). HPLC
analysis showed one large peak and a second smaller
peak of less than one-tenth the area of the large peak.
This is in agreement with previously reported results
using similar analytical methods (Vorages et al. 1982).
The smaller peak is an isomer of bongkrekic acid. The
concentration of the standard was con rmed using its
published molar extinction coe cient ( e 267 36 700;
Lijmbach et al. 1970, 1971).
Growth medium
A modi cation of Rich Coconut Media (RCM) used
in previous studies (Ko 1985) was prepared by blend-
ing 100 g of fresh coconut meat and 150 mL deionized
water 65 C for 3 min at high speed in a Waring
blender. The material was strained through two layers
of cheesecloth until 150 ml liquid was drained o . The
remaining solids were oven-dried and the lipid com-
ponents extracted to form a defatted RCM (dRCM).
This was accomplished by drying a 1 cm thick layer of
65
RCM at 60 C for 12 h. The dried RCM was extracted
by Soxhlet (Association of O cial Analytical Chem-
ists 1990, Nielsen 1994) using diethyl ether at a rate of
4 5 drops per second for 4 h. The dRCM was dried at
60 C for 14 h.
In each experiment, 0.8 g of dried dRCM was used.
Lipid was added to the material by dissolving the
desired lipid in 10 ml ethyl ether and stirring into the
dried dRCM. The beaker in which the lipid was
dissolved was rinsed several times with small volumes
of fresh ether that were added to the dRCM. The
ether was evaporated with occasional mixing for
2 h.
Distilled water (4.1 ml) was added to bring it back to
approximately the moisture content of the original
RCM. All media samples were placed in screw-top
plastic jars and autoclaved at 115 C for 20 min.
Inoculation
The inoculation culture was grown in sterile nutrient
broth in a shaker ask for 48 h at 30 C, with shaking
(250 rotations/min) and 0.9 ml of this culture used to
inoculate each individual experiment resulting in an
initial cell density of approximately 9 108 cfu/g (wet
weight). The screw-top jars containing the media and
the culture were shaken vigorously to distribute the
culture evenly.
The cell density of the inoculation was measured
using a Mannostat photoelectric colorimeter at 520
580 nm. A standard curve of cell density versus
absorbance was prepared using aerobic plate counts
of B. cocovenenans over a range of cell densities
(Collins et al. 1989). The cell density of an inoculation
was estimated from this curve.
Incubation
Inoculated dRCM was incubated at 30 C, 100%
relative humidity, in darkness for 5 days. Samples
were shaken brie y once per day to encourage homo-
geneous growth and to improve oxygen distribution
throughout the medium.
 66 R. A. Garcia et al.

Determination of nal cell concentration
After 5 days, 1 g of the spent medium was aseptically
transferred into a dilution bottle containing 99 ml
sterile phosphate bu er. The bottle was shaken for
30 s and serial dilutions made in sterile phosphate
bu er over the range of 1:100 to 1:109. From each
dilution, 100 m l aliquots were transferred, in duplicate,
to Plate Count Agar. The liquid spread uniformly
over the surface of the agar. All plates were incubated
at 30 C for 36 h and then examined. The number of
colonies on each plate was counted, and plates having
between 25 and 250 colonies were used to determine
the cell concentration.
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lter. Peak areas were recorded using a Hewlett-
Packard 3390A Integrator. An Applied Biosystems
ODS Spheri-5 Reverse Phase Column
(22 cm 4.6 mm i.d. ) was used along with an Applied
Biosystems RP-18 Guard Column. Bongkrekic acid
was eluted in 6.5 min using methanol-water-acetic
acid (80:19:1) mixture and a ow rate of 1.0 ml/min.
The concentration of bongkrekic acid in the media
(dry weight basis) was calculated by comparing peak
areas against the external standard.
Results and discussion

Extraction of bongkrekic acid The addition of 0 50% (dry basis) coconut fat which
had been previously extracted from the same RCM,
back to dRCM did not a ect the growth of B.
Approximately half of the material from an experi- cocovenants which grew to the same cell densities in
ment was spread on a watch glass and dried for 3 h in all experiments (table 1). However, the amounts of
an oven at 70 C. The lipid was extracted by the bongkrekic acid produced varied widely. Bongkrekic
AOAC Soxhlet procedure 920.39C (Association of acid was not detectable with 0 or 10% coconut fat
O cial Analytical Chemists 1990, Nielson 1994). The even though the cell density was > 1010 cfu/g (wet
Soxhlet apparatus was lled with 120 ml diethyl ether weight; table 1). At 20% fat, a small amount
and run at a rate of 4 5 drops per second for 4 h. The
ether from the Soxhlet extraction was concentrated to (0.054 mg/g dry weight) of bongkrekic acid was
approximately 10 ml using a rotary evaporator and formed. As the level of coconut fat was raised from
then transferred to a conical test tube. A slow stream 20 to 40% , the amount of bongkrekic acid produced
of nitrogen was passed over the surface of the liquid increased rapidly to 1.3 mg/g dry weight. Increasing
until its volume was reduced to 2 ml. Methanol (2 ml) the coconut fat from 40 to 50% resulted in a relatively
was added to the ether and the stream of nitrogen was small increase in bongkrecic acid production. The
continued until the volume had again reduced to 2 ml. maximum concentration of bongkrekic acid reached
The extract was then brought to 10 ml by the addition was 1.4 mg/g. This concentration of bongkrekic acid
of methanol. This liquid was transferred to a brown is similar to that reported in previous work (Ko
glass vial and stored ( 20 C) until analysis. 1985).
To test the e ciency of this method, a recovery study
was conducted in which dRCM was spiked with a Table 1. Growth of B. cocovenenans and formation of
known quantity of bongkrekic acid. This spiked bongkrek acid in dRCMa with increasing amounts of
dRCM was extracted and analysed by the same pro- coconut fat.
cedure as unknowns. Approximately 93% of the
original bongkrekic acid was recovered. % coconut fat BA concentration Cell density
(w/w dry) (mg/g dry wt) (cfu/g wet wt)
0 ND 3. 5 1010
10 ND 2. 4 1010
Analysis of bongkrekic acid concentration 20 0.05 5. 2 1010
30 0.49 4. 0 1010
40 1.26 4. 2 1010
Bongkrekic acid extracts were analysed by a modi - 50 1.40 6. 2 1010
cation of the HPLC method of Vorages et al. (1982). ND not detectable.
A Beckman model 110B pump was used along with a Limit of deteciton, 0.001mg/g.
Beckman 160 detector that was tted with a 254 nm a
Defatted Rich Coconut Medium (dRCM).
 E ect of lipids on bongkrekic acid toxin production 67

Table 2. Growth of B. cocovenans and formation of Table 3. Growth of B. cocovenenans and formation of
bongkrek acid in dRCMa containing 3.31mmol free bongkrek acid in dRCMa containing increasing amounts
fatty acid per g of dCRM. of coconut fat.
Concentration Final cell % lauric acid BA concentration, Cell density,
BA concentration (dry wt) mg/g (dry wt) cfu/g (wet wt)
Fatty acid (mg/g dry wt) (cfu/g wet wt)
0 < 0.003 3. 5 1010
Caproic 6: 0 < 0.003
4
< 10 10 < 0.003 2. 7 1010
Caprylic 8: 0 < 0.003 1. 4 108 20 < 0.003 2. 5 1010
Capric 10: 0 < 0.003 2. 4 108 30 < 0.003 9. 4 1010
Lauric 12: 0 0.30 2. 6 1010 40 0.30 2. 6 1010
Myristic 14: 0 0.50 3. 5 1010 50 0.64 4. 2 1010
Palmitic 16: 0 0.20 9. 0 1010 a
Stearic 18: 0 2. 1 1010 Defatted Rich Coconut Medium (dRCM).
< 0.003
Oleic 18: 1 2.62 7. 9 109
Linoleic 18: 2 0.22 3. 0 109
Linolenic 18: 3 0.23 1. 0 1010 support the production of detectable amounts of
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Arachidonic 20: 0 < 0.003
No fat < 0.003 3. 5 1010
a
Defatted Rich Coconut Medium (dRCM).
A series of saturated fatty acids was added to dRCM
in order to determine the e ects of chain length on
bongkrekic acid formation. In each experiment
3.3 mmol of saturated free fatty acid was added to
dRCM. Both caproic (6:0) and arachidonic (20:0)
acids were toxic to B. cocovenenans and within 1 h
after inoculation reduced the viable cell concentration
to less than 104 cfu/g (the limit of detection). The cell
density remained too low to count at the end of the
incubation period. Caprylic (8:0) and capric (10:0)
acids allowed the culture to remain near the incula-
tion density of 108 cfu/g. The remaining fatty acids,
lauric (12:0), myristic (14:0), palmitic (16:0), and
stearic (18:0), supported cell growth; all samples had
cell densities of 2.1 9.0 1010 cfu/g. Only three of the
saturated fatty acids stimulated the production of
detectable amounts of bongkrekic acid. Myrstic acid
(14:0) had the strongest e ect, followed by lauric acid
(12:0), and palmitic acid (16:0) (table 2). These fatty
acids make up 71.5 74.5% (by weight) of the fatty
acids in coconut oil. The other fatty acids tested did
not support the production of detectable amounts of
bongkrekic acid.
Bongkrekic acid is highly unsaturated and has seven
carbon carbon double bonds. It was of interest to
investigate whether the degree of unsaturation of the
fatty acid substrate had an e ect on bongkrekic acid
production. Equimolar amounts of four 18-carbon
fatty acids having di erent degrees of saturation were
added to dRCM and the growth and production of
bongkrekic acid monitored. Stearic (18:0) did not
4
< 10 bongkrekic acid (table 2). Oleic (18:1) acid, however,
supported the production of the largest amount of
bongkrekic acid (2.62 mg/g) seen in any experiment.
Linoleic (18:2) and linolenic (18:3) supported the
production of only small amounts of toxin. Growth
of the organism was seen in all cases.
Lauric acid makes up 45 51% (by weight) of the fatty
acids in coconut fat and was chosen to study the e ect
of fatty acid concentration (Hilditch and Williams
1964). Preliminary experiments indicated that B.
cocovenenans produces bongkrekic acid on media in
which lauric acid is the sole lipid. Experiments were
conducted in which the growth medium contained
between 0 and 50% lauric acid by dry weight. With 0
20% lauric acid, no detectable amount of bongkrekic
acid was produced (table 3). With 30% lauric acid, a
small but detectable amount of the toxin was pro-
duced. Bongkrekic acid production rose steadily as
the amount of lauric acid was increased from 30 to
50% (table 3). The amount of toxin produced on 50%
lauric acid (0.64 mg/g) was lower than the amount
produced on 50% coconut fat (1.4 mg/g). In all
experiments, the cells grew to the same density of
> 1010 cfu/g. This con rms that cell growth is not
necessarily accompanied by bongkrekic acid produc-
tion.
Fat and fatty acid type and concentration were
critical factors in determining the amount of bong-
krekic acid produced in culture. The di erences be-
tween the lowest and highest amounts of bongkrekic
acid formed were > 1400-fold (table 1); > 873-fold
(table 2); and > 213-fold (table 3), all in a dose
dependent manner. In experiments involving varied
levels of coconut fat, toxin was not detected when the
media contained less than 20% fat. This supports the
anecdotal evidence that tempe bongkrek made from
 68 R. A. Garcia et al.
coconut presscake does not become toxic. Coconut
presscake contains 0.53 10% residual fat (depending
on method of oil extraction) and thus would not be
expected to support bongkrekic acid production
based on our experimental data. Because coconut
milk residue is typically used by individuals at home
to make tempe bongkrek, the per cent fat in this
material is not well de ned. However, our data imply
that the lipid content of coconut milk residue is
greater than 20% in cases where toxic tempe bong-
krek was produced. This agrees with data indicating
that the lipid content of coconut milk residue is higher
than coconut presscake (Shurtle and Aoyagi 1997).
The type and concentration of fatty acids in the
growth medium also in uenced bongkrekic acid pro-
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duction. Experiments involving di erent concentra-
tions of lauric acid demonstrated a relationship
between increasing fatty acid concentration and in-
creasing bongkrekic acid production. Saturated fatty
acids of di erent chain lengths are not equivalent in
their ability to stimulate bongkrekic acid production.
Of the saturated fatty acids common in foods, only
lauric (12:0), myrisic (14:0) and palmitic (16:0) acids
support biosynthesis of detectable amounts of toxin.
B. cocovenenans seems particularly well adapted to
utilize coconut oil for bongkrekic acid synthesis be-
cause these three fatty acids make up the majority of
the fatty acids in coconut oil.
The level of unsaturation of fatty acids is also an
important factor in determining the amount of
bongkrekic acid produced. Oleic acid (monounsatu-
rated) stimulated the production of more bongkrekic
acid than any other substrate.
These results show that bongkrekic acid is not likely
to be produced on foods with low to moderate fat
contents. However, this does not necessarily show
that these foods will not be implicated in food poi-
soning caused by B. cocoveneanans. Maize kernals
average 3.9% fat (Eckey 1954). Fermented corn meal,
which is made from ground maize, has caused
poisonings and has been found to be highly contami-
nated with B. cocovenenans. In these cases, the poi-
soning may be caused by toxo avin and not
bongkrekic acid. In our work, all media that sup-
ported growth of B. cocovenenans were yellow sug-
gesting the presence of toxo avin even when
bongkrekic acid was absent. Alternatively, it may be
that the high 18:1 content of corn oil results in
bongkrekic acid production even though the lipid
content is well below that found necessary for bong-
krekic acid formation in our work. We are currently
investigating this possibility as well as the factors
a ecting toxo avin formation in corn meal.
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