Makalah Tempe Bongkrek : Asam Bongkrek dan Toksoflavin)

PENGARUH ASAM BONGKREK DAN TOKSOFLAVIN PADA TEMPE
BONGKREK TERHADAP KESEHATAN
Dosen Pengampu : Dra. Setyorini Sugiastuti, M.Si., Apt.
Disusun oleh:
Nama : Nesha Mutiara
NPM : 2017210155
Kelas : Analisis Makanan D
FAKULTAS FARMASI
UNIVERSITAS PANCASILA
JAKARTA
2019

KATA PENGANTAR
Puji syukur kepada Tuhan yang Maha Esa atas kuasa-Nya sehingga penulis dapat
menyelesaikan makalah yang berjudul “Pengaruh Asam Bongkrek dan Toksoflavin pada Tempe
Bongkrek terhadap Kesehatan” sebagai tugas akhir mata kuliah analisis makanan. Pada
kesempatan ini penulis berterima kasih kepada Ibu Dra. Setyorini Sugiastuti, M.Si., Apt. selaku
dosen pengampu mata kuliah analisis makanan yang turut membimbing dalam penyusunan
makalah ini.
Penulis menyadari bahwa makalah ini masih jauh dari sempurna, untuk itu saran dan kritik
yang bersifat membangun sangat diharapkan. Akhir kata, penulis berharap makalah ini dapat
menjadi referensi bagi pembaca untuk meningkatkan kewaspadaan dalam mengonsumsi
pangan khususnya tempe bongkrek.
Penulis

DAFTAR ISI
Halaman
HALAMAN JUDUL
KATA PENGANTAR
DAFTAR ISI
DAFTAR TABEL
DAFTAR GAMBAR
DAFTAR LAMPIRAN
BAB I PENDAHULUAN 1
i. LATAR BELAKANG 1
ii. TUJUAN PEMBUATAN MAKALAH 1
iii. MANFAAT PEMBUATAN MAKALAH 2
BAB II TINJAUAN PUSTAKA 3
i. DESKRIPSI TEMPE BONGKREK 3
ii. DESKRIPSI BAKTERI Pseudomonas Cocovenenans 4
1. Klasifikasi bakteri Pseudomonas cocovenenans 4
2. Morfologi dan fisiologi Pseudomonas cocovenenans 4
3. Penyakit yang ditimbulkan oleh Pseudomonas cocovenenans 5
iii. DESKRIPSI TOKSOFLAVIN 5
iv. DESKRIPSI ASAM BONGKREK 5
v. CARA PEMBUATAN TEMPE BONGKREK 6
vi. IDENTIFIKASI DAN PENETAPAN KADAR ASAM BONGKREK PADA
TEMPE BONGKREK 6
vii. MANIFESTASI KLINIS KERACUNAN ASAM BONGKREK DAN TOKSOFLAVIN 7
BAB III TINJAUAN KHUSUS 8

BAB IV PEMBAHASAN 8
BAB V SIMPULAN DAN SARAN 9
i. SIMPULAN 9
ii. SARAN 9
DAFTAR PUSTAKA 10
DAFTAR TABEL
Halaman
Tabel II. 1 Kandungan nutrisi tempe bongkrek per 100 gram 3
DAFTAR GAMBAR
Halaman
Gambar II. 1 Tempe Bongkrek 3
Gambar II. 2. Pseudomonas cocovenenans 4
DAFTAR LAMPIRAN
Wang, Ma, dan Wang. 1996. Enhanced Vasocontraction of Rat Tail Arteries by Toxoflavin.
British Journal of Pharmacology 117, 293 – 298.
Henderson dan Lardy. 1970. Bongkrekic Acid: An Inhibitor of the Adenine Nucleotide
Translocase of Mitochondria. The Journal of Biological Chemistry Vol. 245, No. 6, Issue of
March 25, pp. 13-19-1326.

1
BAB I
PENDAHULUAN
i. LATAR BELAKANG
Keragaman bahan pangan di Indonesia mempengaruhi varietas jenis makanan
tradisional tiap daerah. Salah satu makanan tradisional yang dibahas dalam
makalah ini adalah tempe bongkrek yang berasal dari Banyumas, Jawa Tengah.
Tempe bongkrek adalah makanan yang terbuat dari kacang kedelai dan ampas
kelapa melalui proses fermentasi. Bentuknya mirip dengan tempe berbahan dasar
kacang kedelai dan jamur Rhizopus sp., namun beberapa tempe bongkrek dapat
berwarna sedikit kuning dan teksturnya lebih keras. Tempe bongkrek inilah yang
dapat menyebabkan keracunan karena kontaminasi bakteri Burkholderia galdioli
atau yang lebih dikenal sebagai Pseudomonas cocovenenans yang menghasilkan
racun toksoflavin dan asam bongkrek. Potensi mematikan tempe bongkrek ini
mendorong pemerintah untuk melarang produksi tempe bongkrek sejak tahun 1969.
Sayangnya, kebijakan pemerintah ini tidak dihiraukan oleh masyarakat. Alhasil, 37
warga Kecamatan Lumbir, Banyumas, tewas pada tahun 1988.
Pseudomonas cocovenenans adalah bakteri aerob fakultatif yang tumbuh subur di
tempat berkadar minyak tinggi, sehingga sering dijumpai dalam tempe bongkrek
apabila ampas kelapa yang digunakan tidak segar. Bakteri ini menghasilkan dua
jenis racun yaitu asam bongkrek dan toksoflavin yang merupakan antibiotik
berwarna kuning cerah yang membunuh jamur Rhizopus sp. Asam bongkrek yang
terakumulasi di dalam tubuh menyebabkan kadar glukosa darah meningkat karena
mobilisasi glikogen dari liver ke otot dan menghambat fosforilasi oksidatif pada
mitokondria liver sehingga dapat membunuh penderita.
Walaupun tempe bongkrek berpotensi mematikan, masyarakat tetap dapat
menikmati tempe bongkrek secara aman dengan menambahkan beberapa bahan
untuk mencegah terbentuknya racun toksoflavin dan asam bongkrek, seperti jamur
Monilla sitophila, garam dapur 1.5 – 2% pada ampas kelapa, dan antibiotik
Aureomycin dan Terramycin [1].
ii. TUJUAN PEMBUATAN MAKALAH
1. Mengetahui kandungan senyawa beracun dalam tempe bongkrek.

2
2. Mengetahui penyebab tempe bongkrek beracun.
3. Mengetahui mekanisme kerja racun dalam tempe bongkrek.
4. Mengetahui cara pembuatan tempe bongkrek yang baik dan benar.
5. Mengetahui bahan – bahan tambahan yang dapat mencegah terbentuknya racun
dalam
tempe bongkrek.
6. Mengetahui karakteristik tempe bongkrek beracun.
7. Mengetahui kandungan gizi per 100 gram tempe bongkrek.
iii. MANFAAT PEMBUATAN MAKALAH
Makalah ini disusun dengan harapan kewaspadaan pembaca dalam memproduksi
dan mengkonsumsi tempe bongkrek meningkat serta pembaca mengetahui dan
memahami cara pembuatan tempe bongkrek yang baik dan benar, mengetahui
bahan – bahan tambahan yang dapat mencegah terbentuknya racun toksoflavin dan
asam bongkrek, mengenali tempe bongkrek beracun, serta mengetahui kandungan
gizi per 100 gram tempe bongkrek.

3
BAB II
TINJAUAN PUSTAKA
i. DESKRIPSI TEMPE BONGKREK
Gambar 1. Tempe Bongkrek
Tempe bongkrek adalah jenis produk fermentasi dari Banyumas, Jawa Tengah
yang dibuat dari kacang kedelai, ragi tempe, dan ampas kelapa. Tempe ini seringkali
menyebabkan keracunan karena terkontaminasi oleh bakteri Burkholderia galdioli
atau yang dikenal sebagai Pseudomonas cocovenenans yang menghasilkan racun
berupa toksoflavin dan asam bongkrek, serta memusnahkan jamur Rhizopus karena
efek antibiotik dari asam bongkrek. Tempe bongkrek terbuat dari ampas kelapa
mengandung minyak dalam kadar yang tinggi dan lebih lembab. Kandungan minyak
yang tinggi berbahaya untuk dikonsumsi karena dapat menjadi tempat pertumbuhan
yang baik bagi bakteri patogen. Kondisi ini tidak akan terjadi apabila ampas kelapa
yang digunakan masih segar [2].
Tabel 1. Kandungan nutrisi tempe bongkrek per 100 gram.
Kandungan nutrisi Jumlah per 100 gram
Energi 119 kkal
Protein 4,4 gram
Lemak 3,5 gram
Karbohidrat 18,3 gram
Kalsium 27 mg
Fosfor 100 mg
Zat besi 3 mg
Vitamin B1 0,08 mg

4
ii. DESKRIPSI BAKTERI Pseudomonas cocovenenans
Gambar 2. Pseudomonas cocovenenans
1. Klasifikasi bakteri Pseudomonas cocovenenans
Kingdom : Bacteria
Phyllum : Protobacteria
Kelas : Beta proteobacteria
Ordo : Burkholderiaceae
Genus : Bulkholderia
Species : B. gladioli
2. Morfologi dan fisiologi Pseudomonas cocovenenans
Pseudomonas cocovenenans berbentuk batang, dapat bergerak (motil)
menggunakan lima silia pada salah satu ujungnya. Bentuk bakteri tersebut dapat
berubah – ubah tergantung pada jenis medium yang digunakan. Pada umumnya
berukuran 0,5 – 1,0 mikrometer x 1,5 – 4,0 mikrometer. Bersifat kemoorganotrof,
yaitu memperoleh energi dari hasil oksidasi senyawa kimia organik. Dapat tumbuh
pada rentang pH 6 – 8 dengan pertumbuhan optimum pada pH 8,0. Pada pH sama
dengan atau lebih rendah dari 6,0 produksi racun terhambat dan pada pH sama
dengan atau lebih rendah dari 5,0 pertumbuhan Pseudomonas cocovenenans
terganggu.

5
Pseudomonas cocovenenans bersifat aerob fakultatif, dapat tumbuh di berbagai
media dan biasanya mengeluarkan zat yang berwarna kuning. Bersifat gram negatif,
bersel tunggal, dan dapat tumbuh pada suhu kamar atau suhu 37O
C. Pseudomonas
cocovenenans timbul karena proses fermentasi yang tidak sempurna sehingga
menghasilkan enzim tertentu yang dapat memecah sisa minyak kelapa dalam tempe
bongkrek melalui proses hidrolisis lipid dari minyak kelapa menjadi gliserol dan asam
lemak. Fraksi gliserol setelah mengalami reaksi – reaksi biokimiawi menjadi
senyawa yang berwarna kuning yang disebut toksoflavin, sedangkan asam
lemaknya khususnya asam oleat menjadi asam bongkrek yang tidak berwarna [3].
3. Penyakit yang ditimbulkan oleh Pseudomonas cocovenenans
Infeksi pada manusia yang khas ialah menyebabkan keracunan toksoflavin dan
asam bongkrek. Toksoflavin menghasilkan hidrogen peroksida yang toksik terhadap
sel. Asam bongkrek memobilisasi glikogen di dalam liver menyebabkan
hiperglikemia lalu hipoglikemia serta menghambat pembentukan ATP yang dapat
menyebabkan kematian [4].
iii. DESKRIPSI TOKSOFLAVIN
Toksoflavin (C7H7N5O2) merupakan pigmen berwarna kuning yang bersifat
fluoresens dan stabil terhadap oksidator. Toksoflavin dapat menyebabkan kematian
karena di dalam tubuh penderita terbentuk hidrogen peroksida dalam jumlah yang
banyak tanpa diimbangi enzim katalase yang cukup dari tubuh. Toksoflavin juga
dapat menyebabkan transpor gula ke dalam eritrosit terhambat sehingga terjadi
hemolisis karena terhambatnya aktivitas enzim glutamat transferase serta alkali
fosfatase dalam eritrosit. Mekanisme kerjanya yaitu toksoflavin membawa elektron
antara NADH dan oksigen yang memungkinkan kerja sitokrom dibuat pintas
sehingga menghasilkan hidrogen peroksida. Reaksi yang terjadi yaitu [5]:
NADH + Toks. A Toks.-H2 _ 2NAD+
Toks.-H2 + O2 a Toks. + H2O2
iv. DESKRIPSI ASAM BONGKREK
Asam bongkrek (C28H38O7) merupakan asam trikarboksilat tidak jenuh yang dapat
mengganggu metabolisme glikogen dengan memobilisasi glikogen dari liver
sehingga terjadi hiperglikemia lalu berubah menjadi hipoglikemia. Asam bongkrek
bekerja secara akumulatif dan menyebabkan kematian mendadak setelah racunnya

6
terakumulasi di dalam tubuh karena tidak mudah diinaktifkan, didetoksifikasi,
maupun diekskresi oleh tubuh. Asam bongkrek bersifat inhibitor kuat bagi
mitokondria karena dapat menutupi gugus –SH dari ATP –ase sehingga produksi
ATP pada mitokondria terhenti, akibatnya ATP diproduksi di luar mitokondria secara
glikolisis dari glikogen cadangan yang terdapat di dalam liver. Proses penguraian
glikogen liver, jantung, dan otot – otot akan menyebabkan kadar glukosa darah
meningkat, lalu persediaan glikogen habis. Dalam waktu singkat kadar glukosa
darah segera menurun dan menyebabkan penderita mengalami asidosis [6].
v. CARA PEMBUATAN TEMPE BONGKREK
Proses pembuatan tempe bongkrek membutuhkan bahan berupa kacang kedelai,
ampas kelapa, ragi tempe, dan air. Langkah – langkah pembuatannya yaitu:
1. Kacang kedelai dipilah dan dibersihkan dengan air mengalir
2. Kacang kedelai dipipil lalu direndam dengan air bersih selama 36 jam
3. Kacang kedelai dikukus dan ampas kelapa dikeringkan
4. Kacang kedelai ditiriskan
5. Kacang kedelai dicampur dengan ampas kelapa yang telah kering
6. Ditambahkan ragi atau inokulasi pada campuran kacang kedelai dan ampas
kelapa
7. Disimpan pada suhu ruang
vi. IDENTIFIKASI DAN PENETAPAN KADAR ASAM BONGKREK PADA
TEMPE BONGKREK
Asam bongkrek pada tempe bongkrek dapat diidentifikasi menggunakan test kit
komersiall seperti Biologic NG2 System. Metode yang paling umum digunakan yaitu
16S rDNA sequencing, tetapi kadang kala dapat memberikan hasil yang tidak
akurat. Metode lain yang dapat digunakan yaitu capillary electrophoresis – single
strand conformation polymorphisms (CE-SSCP), microarray analysis, atau probe-
based cell fishing. Metode yang paling dapat diandalkan adalah multiplex PCR
protocol [7].
Keberadaan dan kadar asam bongkrek pada sampel lingkungan dapat
diidentifikasi dan ditetapkan menggunakan liquid thin layer chromatography,

7
chromatography – mass spectroscopy, dan high pressure liquid chromatography
(HPLC). Asam bongkrek dan toksoflavin tidak boleh ada sedikit pun pada tempe
bongkrek [8].
vii. MANIFESTASI KLINIS KERACUNAN ASAM BONGKREK DAN
TOKSOFLAVIN
Periode laten setelah paparan asam bongkrek dan toksoflavin dari makanan
terkontaminasi bakteri Pseudomonas cocovenenans dilaporkan satu hingga sepuluh
jam kemudian. Target organ utama yaitu liver, otak, dan ginjal. Tanda dan gejala
pada manusia mirip dengan penemuan klinis dari racun pada mitokondria lainnya,
namun lebih variatif dan waktu latensinya lebih lama. Gejala yang dilaporkan yaitu
malaise, pusing, keringat berlebih, palpitasi, nyeri abdominal, muntah, diare,
hematuria, dan retensi urinaria. Penemuan selama pemeriksaan pasien meliputi
hipotensi, aritmia, hiperthermia, ikterus, jaundice, letargi, delirium, syok, bahkan
dapat koma dan berujung kematian. Kematian dapat terjadi sejak satu hingga dua
puluh jam setelah kemunculan tanda dan gejala. Abnormalitas yang terdeteksi
selama pemeriksaan di laboratorium meliputi inisial hiperglikemia, fungsi liver yang
abnormal, dan peningkatan jumlah leukosit [9].
Hanya dengan mengonsumsi 1 – 1,5 mg tempe bongkrek beracun dapat
menyebabkan kematian. Tingkat gejala keracunan tempe bongkrek yaitu:
1. Ringan : pusing, mual, dan muntah.
2. Sedang : pusing, mual, muntah, dan sakit perut.
3.Berat : diare, kejang, keluar buih putih dari mulut.
4. Meninggal : ada bercak darah beku di bawah kulit.

8
BAB III
TINJAUAN KHUSUS
Kejadian Luar Biasa (KLB) akibat kontaminasi bakteri Pseudomonas cocovenenans pada
tempe bongkrek hanya terjadi di Indonesia. Kasus keracunan pertama kali dilaporkan pada
tahun 1895. KLB terulang kembali sejak tahun 1975, mengakibatkan hampir 3000 kasus
keracunan asam bongkrek meliputi setidaknya 150 kematian [7]. Di Indonesia, rata – rata
kematian yang dilaporkan mencapai 60% diakibatkan oleh keracunan asam bongkrek. KLB
ketiga terjadi pada tahun 1988 yang memakan 34 korban di Kecamatan Lumbir, Banyumas,
sejak itu produksi tempe bongkrek dilarang pemerintah, tetapi kasus keracunan akibat
produksi secara diam – diam masih berlanjut [10].
BAB IV
PEMBAHASAN
Berdasarkan studi literatur dari jurnal, buku, dan artikel yang relevan dengan topik
makalah, diperoleh informasi bahwa tempe bongkrek yang dibuat dari ampas kelapa
yang tidak segar dapat terkontaminasi oleh bakteri patogen Pseudomonas
cocovenenans. Bakteri tersebut menghasilkan senyawa beracun yaitu asam
bongkrek dan toksoflavin yang dapat menyebabkan kematian. Senyawa beracun ini
menyerang mitokondria korban sehingga mengganggu proses metabolisme tubuh
korban. Kasus keracunan massal yang disebut Kejadian Luar Biasa (KLB) akibat
tempe bongkrek beracun pertama kali dilaporkan pada tahun 1895, terulang kembali
pada tahun 1975, dan 1988. Sejak KLB pada tahun 1988, pemerintah melarang
produksi tempe bongkrek, namun produksi secara diam – diam tetap berlanjut.
Tempe bongkrek dapat aman dikonsumsi jika dalam proses pembuatannya
menggunakan bahan – bahan yang segar terutama ampas kelapa yang tidak terlalu
berminyak.
Walaupun tempe bongkrek berpotensi mematikan, masyarakat tetap dapat
menikmati tempe bongkrek secara aman dengan menambahkan beberapa bahan
untuk mencegah terbentuknya racun toksoflavin dan asam bongkrek, seperti jamur
Monilla sitophila, garam dapur 1.5 – 2% pada ampas kelapa, dan antibiotik
Aureomycin dan Terramycin. Penggunaan Aureomycin dan Terramycin dalam
produksi tempe bongkrek tidak dianjurkan karena dikhawatirkan dapat menyebabkan
konsumen mengalami resistensi antibiotik Aureomycin dan Terramycin.

9
BAB V
SIMPULAN DAN SARAN
i. SIMPULAN
1. Tempe bongkrek merupakan makanan tradisional khas Banyumas, Jawa Tengah
yang dibuat dari ampas kelapa.
2. Tempe bongkrek dapat beracun jika terkontaminasi bakteri patogen
Pseudomonas cocovenenans yang menghasilkan senyawa beracun asam bongkrek
dan toksoflavin.
3. Tempe bongkrek beracun dapat menyebabkan kematian sehingga asam
bongkrek dan toksoflavin tidak boleh terdapat pada tempe bongkrek.
4. Tempe bongkrek beracun memiliki karakteristik berwarna kuning dan berbau tidak
sedap.
5. Tempe bongkrek dapat dibuat secara aman dengan menambahkan beberapa
bahan untuk mencegah terbentuknya racun toksoflavin dan asam bongkrek, seperti
jamur Monilla sitophila, garam dapur 1.5 – 2% pada ampas kelapa, dan antibiotik
Aureomycin dan Terramycin
ii. SARAN
Sebaiknya masyarakat lebih berhati – hati jika hendak membuat dan
mengonsumsi tempe bongkrek serta memperhatikan karakteristik fisik tempe
bongkrek sebelum mengonsumsinya untuk menghindari keracunan asam bongkrek
dan toksoflavin.

10
DAFTAR PUSTAKA
1.http://blog.sivitas.lipi.go.id/blog.cgi?isiblog&1253162990&&&1036006250&&14418
55384&rhar003& (diakses pada 25 Maret 2019 pukul 18.26 WIB)
2. Shurtleff dan Aoyagi. 1979. The Book of Tempeh. London: Harper.
3. Garcia, R.A. 1999. The effcet of lipids on bongkrekic acid toxin production by
Burkholderia cocovenenans in coconut media. Food Additives and Contaminants,
volume 16, 63 – 69.
4. Stern, K.G. 1934. Oxidation – Reduction Potentials of Toxoflavin. Journal of
Biochemistry, 500 – 508.
5. Van, D. 1960. On Toxoflavin, the Yellow Poison of Pseudomonas cocovenenans.
Rec. Trav. Chim., volume 799, 255.
6. Henderson, P.J. 1970. Bongkrekic Acid. Journal of Biochemical Chemistry,
volume 245, 1319 – 1326.
7. Lynch KH, Dennis JJ. Burkholderia. In: LiuD, editor. Molecular detection of
foodborne pathogens. Boca Raton: CRC Press; 2009. Pp. 331-343.
8.Hu WJ, Zhang GS, Chu FS, Meng HD, Meng ZH. Purification and partial
characterization offlavotoxin A. Appl Environ Microbiol. 1984;48(4):690-693.
9. Cox J, Kartadarma E, Buckle KA. Burkholderia cocovenenans. In:Hocking AD,
editor. Foodborne microorganisms of public health significance. 6th
. Sydney:
Australian Institute of Food Science & Technology; 1997. Pp. 521-530.
10. Arbianto P. Bongkrek food poisoning. In: Java: Proceedings of the Fifth
International Conference on Global Impacts of Applied Microbiology; 1979. Pp. 371-
4.

THE Jonxi-i~~ OF BIOLOGICAL CHEMISTRY
Vol. 245, No. 6,Issue of March 25, pp. 1319-1326,197O
Printedin U.S.A.
Bongkrekic Acid
AN INHIBITOR OF THE ADENINE NUCLEOTIDE TRANSLOCASE OF MITOCHONDRIA*
(Received for publication, November 3, 1969)
PETER J. F. HENDERSONI AND HENRY A. LARDY
From the Institute for Enzyme Research,University of Wisconsin, Madison, Wisconsin 53706
SUMMARY
The antibiotic bongkrekic acid is shown to inhibit the phos-
phorylation of added ADP coupled with either the reactions of
the respiratory chain or the dismutation of oc-ketoglutarate in
mammalian mitochondria. It also prevents the utilization of
ATP for energy-linked cation transport and in energy dissipa-
tion by uncoupling agents. However, bongkrekic acid does
not inhibit the Mg++-requiring ATPase of submitochondrial
particles, the phosphorylation of endogenous ADP, or the
stimulation of oxidation caused by arsenate. The ADP en-
hancement of the arsenate effect is prevented. These results
distinguish its site of action from that of oligomycin (rutamy-
tin), and indicate that bongkrekic acid inactivates transloca-
tion of adenine nucleotides into mitochondria. This is con-
firmed by measurement of the uptake of W-labeled adenine
nucleotides. The extent of inhibition of oxidative phos-
phorylation by bongkrekic acid shows a sigmoidal relation-
ship to the concentration of antibiotic, which is in contrast to
the linear relationship reported for atractyloside, a known
inhibitor of the translocase enzyme.
Bongkrekic acid is one of the toxic principles produced by the
organism Pseudomonas cocovenenans and was first isolated by van
Veen and Mertens (1). The appearance of this organism on the
coconut food product “bongkrek” in Indonesia led to outbreaks
of fatal food poisoning, the main symptom being a strong
hyperglycemia followed by hypoglycemia and death (2, 3).
Chemical studies of bongkrekic acid (3, 4) have recently led to
the elucidation of its structure (5), which is reproduced in Fig. 1.
Investigations of Welling, Cohen, and Berends (6) established
that bongkrekic acid is an inhibitor of oxidative phosphorylation
in rat heart mitochondria, and that, unexpectedly, it also ac-
celerated the oxidation of succinate and /3-hydroxybutyrate.
The latter result may have been due to the presence of fatty
acids in the bongkrekic acid preparation, as suspected by these
* This work was supported by grants from the National Science
Foundation, the National Institutes of Health, and the Life
Insurance Medical Research Fund. This is the 14th paper in the
series. “Antibiotics as Tools for Metabolic Studies.” For the
previous paper in this series see E. Dorschner and H. Lardy, Anti-
microbical agents and chemotherapy, 1968, p. 11.
$ Recipient of a Fulbright Travel Fellowship.
workers and confirmed in a communication to us from Dr. G. W.
M. Lijmbach (see Reference 5). The recent availability of a
pure preparation prompted us to reinvestigate these effects, and
it has been confirmed that bongkrekic acid inhibits oxidative
phosphorylation in mitochondria from rat liver but it does not
appear to accelerate oxidation of any of the normal substrates.
A number of other antibiotics are known to inhibit oxidative
phosphorylation; these include the oligomycins (7-ll), aurovertin
(8, 9), and atractyloside (12, 13). The oligomycins appear to
act at the level of the mitochondrial enzymes responsible for
coupling electron transport to phosphorylation, the so-called
Fo-Fl complex (14-19), whereas aurovertin interacts with F1
(19). Atractyloside acts by preventing access of adenine
nucleotides to enzymic sites within the mitochondria, probably
by inhibition of a specific translocase (20-27). These investiga-
tions of the site susceptible to atractyloside have established
many criteria by which an inhibition of the coupling process may
be distinguished from an inhibition of adenine nucleotide trans-
location, although both may have as an end result the loss of
oxidative phosphorylation. The criteria will be applied in the
light of the observations below to show that bongkrekic acid
also prevents adenine nucleotide translocation.
Atractyloside and bongkrekic acid have very different chemical
structures (see Fig. 1 and Reference 49). It is therefore not
surprising that the kinetics of bongkrekic acid inhibition differ
from those induced by atractyloside and support the suggestion
of Vignais et al. (28) and evidence of Winkler and Lehninger (29)
that the translocase is an “allosteric” enzyme.
EXPERIMENTAL PROCEDURES
Rat liver mitochondria were prepared as described elsewhere
(30), except that a medium of 250 mM sucrose, 4 mM Tris-Cl,
t--2,
82
Bongkrekic Acid
FIG. 1. The structure of bongkrekic acid (taken from Refer-
ence 5).
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Bongkrekic Acid Inhibition of Nucleoticle Translocation Vol. 245, No. 6
and 1 mM Tris-EGTA (final pH 7.4) was used throughout the
centrifugation procedures, and also for the final suspension
of mitochondria. Submitochondrial particles were prepared by
treating mitochondria with the detergent Lubrol W-X (29) or
by sonic disruption (31). In both cases the treated suspensions
were freed of whole mitochondria by centrifugation at 20,000 x
g for 10 min, and the resulting supernatant fraction was centri-
fuged at 93,000 x g for 45 min to bring down the submito-
chondrial particles. These were then resuspended in a small
volume of 250 mM sucrose, 4 mM Tris-Cl, pH 7.4, lightly ho-
mogenized, and used in the amounts indicated in the legends to
the figures.
Oxygen consumption and P:O ratios were measured either by
conventional manometic techniques or by utilizing collodion-
covered vibrating platinum electrodes supplied with the Gilson
oxygraph (32) or Aminco-Chance dual wave length spectropho-
tometer. The latter instrument and also the Aminco-Bowman
spectrophotofluorometer were used to follow oxidation-reduction
of intramitochondrial pyridine nucleotides. Simultaneous
measurements of alkali metal cation and proton movements,
oxygen uptake, and light scattering (volume changes) of mito-
chondrial suspensions were performed with the apparatus
developed by Pressman (33). In this case a slow stream of
nitrogen was directed over the open cuvette to reduce 02 diffusion
into the mitochondrial suspension. ATPase activity was
measured by the procedure of Lardy and Wellman (34) with the
use of the Pi assay of Sumner (35) ; mitochondrial protein, by a
biuret method (36); and a-ketoglutarate, by the method of
Friedemann (37).
Uptake of 8-14C-labeled XDP (Schw-arz BioResearch) or ATP
(Amersham-Scarle) into mitochondria was assayed by incubating
the mitochondria in 110 mM KCl, 30 mM Tris-Cl (pH 7.4), and
1 mM Tris-EDTA enriched with the labeled nucleotide (0.2 to
0.5 PCi per pmole) at the concentrations to be indicated. Each
sample, usually 300 ~1 in volume, was contained in a micro,
plastic, centrifuge tube. After 20 min, the tubes were spun for
2 min on the Beckman microfuge to separate the mitochondria.
An aliquot of each supernatant fluid was taken and counted in
18 ml of Bray’s scintillation fluid (38) by means of a Packard
Tri-Carb scintillation spectrometer, model 3310, and the re-
mainder of the supernatant was carefully separated from the
pellet. The walls of the tubes were dried with absorbent swabs
and 0.3 ml of 91% formic acid was added and mixed with the
pellet, which was allowed to solubilize overnight. Aliquots were
then taken for counting. Variations between duplicate experi-
ments and between nucleotide uptake from supernatant fractions
and appearance in the pellets were always less than 3%.
Maferials-Bongkrekic acid was kindly donated by Dr. G. W.
RI. Lijmbach and Dr. W. Berends, of the University of Delft,
Holland. Rutamycin was obtained from Eli Lilly and Company,
and atractyloside, originally from Dr. A. Bruni, through the
courtesy of Dr. D. W. Allmsn, University of Wisconsin. Anti-
biotic X-53’i12 was supplied by Dr. Julius Berger, Hoffmann-
LaRoche, Lubrol-WX by ICI Chemicals, Providence, Rhode
Island.
RESULTS
Inhibition of Phosphorylation of ADP
When added to coupled mitochondria respiring in the presence
of inorganic phosphate, i.e. State 4 (39), ADP causes an ac-
celeration of electron transport and oxidation of endogenous
NAD(P)H, which revert to the initial rates when all the ADP is
converted to ATP (Fig. 2). Bongkrekic acid (2.7 mpmoles per
mg of protein) prevented the respiratory chain phosphorylation,
measured by these criteria (Fig. 2). The effect is not due to
inhibition of electron transport, since subsequent addition of
uncoupling agents induced maximal rates of respiration. Similar
results were obtained measuring phosphorylation coupled to
oxidation of succinate, ol-ketoglutarate, glutamate, P-hydroxy-
butyrate, glutamate + malate, pyruvate, proline, or isocitrate.
At levels of inhibition less than 50%, both phosphorylation
and respiration were prevented to the same extent; thus, the
P:O ratio remained constant. Half-maximal inhibition oc-
curred at about 0.30 m~mole of bongkrekic acid per mg of
protein with succinate as substrate.
Fig. 3 shows that the substrate level of phosphorglation
associated with the anaerobic dismutation of ol-ketoglutarate
(4042) is also prevented by bongkrekic acid; the higher concen-
trations caused a small but reproducible decrease in the consump-
tion of c+ketoglutarate, which may reflect an impairment of the
dehydrogenase enzyme (see Reference 6). The mechanism of
the apparent uncoupling depicted in Fig. 3 will be considered
under “Discussion.”
In other experiments rutamycin at concentrations up to 2.5 pg
per mg of mitochondrial protein did not affect either l’i or
oc-ketoglutarate disappearance.
It may be concluded that bongkrekic acid impairs both the
respiratory chain and the substrate level phosphorylation
processes, confirming the results of Welling et al. (6). Sillce
uncoupling agents relieve the inhibition of respiration caused
by bongkrekic acid, and since the respiratory chain is not
involved in the phosphorylation accompanying the anaerobic
dismutation of a-ketoglutarate, the effect of bongkrekic acid is
not mediated by an inhibition of the electron transfer reactions.
The pure sample of bongkrekic acid used for t’hese experi-
ments did not accelerate the oxidation of succinate or fl-hy-
droxybutyrate; it is therefore probable that the acceleration
obtained by Welling et al. (6) resulted from contamination of
the bongkrekic acid by fatty acids which are known to bc potent
uncouplers of mitochondrial oxidations (43, 44).
Inhibition of 11TP-requiring Mitochondrial Reactions
Cation Transport-Fig. 4 shows that bongkrekic acid reversed
the potassium uptake and swelling of mitochondria induced by
valinomycin (45) when XTP was the source of energy. The
cessation of H+ production indicated that the hydrolysis of
,4TP had ceased. It did not prevent these effects when energy
was provided by oxidation of succinate (Fig. 4) or other osidiza-
ble substrates. Table I shows that bongkrekic acid also pre-
vented hydrolysis of ATP when mitochondria were treated with
other ionophorous antibiotics, namely, gramicidin (45)) mon-
actin, dinactin (46), and monazomycin (47) ; in every case
bongkrekic acid totally inhibited the consumption of ATP.
Bongkrekic acid itself did not induce cation transport either into
or out of the mit,ochondria.
Uncoupler-accelerated :I TPase-The mitochondrial ATPase
activity is believed to represent a reversal of the reactions w!uch
lead to ATP synthesis from hDP + Pi (34, 42). The rate of
the ATP breakdown is thus greatly enhanced by uncoupling
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Issue of March 25, 1970 P. J. F. Henderson and H. A. Lardy 1321
BKA CICCP
TIME (MINI
I I I I I
0 I.0 2.0
mpMOLES BKA/mq. PROTEIN
FIG. 2 (left). Inhibition of oxidative phosphorylation by bong-
krekic acid (B&l). Mitochondria (15 mg of protein) were
suspended in a l-cm light path cuvette containing 3 mM triethyl-
aminomalate, 3 m triethylaminoglutamate, 2 ells triethylamino-
Pi, 9 rnM triethylamino-Cl, 14 mM KCl, and 190 mM sucrose, pH 7.4.
The temperature was 21” and the final volume 2.5 ml. NAD (P)H
was measured by differential absorbance at 340 and 374 rnp (the
calibration represents a transmission difference of 7%). Addi-
tions were 0.4 mM Tris-ADP, 0.016 mM bongkrekic acid and 2 X
10-r M carbonyl cyanide m-chlorophenylhydrazone (CZCCP).
FIG. 3 (right). Inhibition by bongkrekic acid of phosphoryla-
tion accompanying the anaerobic dismutation of a-ketoglutarate
(&G). Portions (0.2 ml) of mitochondria (10.6 mg of protein)
were mixed with 1.8-ml portions of medium (30”) containing 4.7
mM triethylamino-or-ketoglutarate, 4.7 mM NH&l, 1 m sodium-
ADP, 1.9 mM triethylamino-Pi, 2 mM MgClg, 37.5 mM KCI, 37.5 ells
KF, 18 mM Tris-Cl, and 70 mM glucose, pH 7.4. Antimycin (4 rg)
and rotenone (3 mpmoles) were present to inhibit respiration; 3
mg of yeast hexokinase to complete the Pi acceptor system, and
bongkrekic acid as indicated (an equal volume of the ethanol sol-
vent was present in each). After 25 min, the reaction was ter-
minated by the addition of trichloracetic acid, and the Pi and
ol-ketoglutarate remaining were estimated.
agents, and Table I indicates that this enhancement was pre-
vented by bongkrekic acid with all the uncoupling agents tested.
The results so far described suggested three possible loci sus-
ceptible to inhibition by bongkrekic acid. It could inactivate
the factors responsible for coupling electron transport to oxida-
tive phosphorylation; it could prevent access of adenine nucleo-
tides to mitochondrial enzymes contained within the cristae;
or it could prevent the uptake of Pi required for oxidative phos-
phorylation, and also prevent its egress, thus inhibiting the
ATPase via a mass action effect. The first two possibilities are
now examined by comparing bongkrekic acid with the antibiotic
inhibitors rutamycin (10) and atractyloside, noting that its
inhibition of the anaerobic dismutation already distinguishes
bongkrekic acid from rutamycin.
Inhibition of ATPase Activity of Submitochondrial Particles
Submitochondrial particles prepared by sonic disruption or
treatment with Lubrol W-X possess a Mg*-activated ATPase.
Unlike whole mitochondria, these particles appear to have the
ATPase on the external surface of the particles, so that there
is no membrane barrier between the ATP substrate and the
enzyme (29). Table II shows that this ATPase is susceptible
to inhibition by rutamycin, but not by atractyloside or bong-
krekic acid, when the particles are made by either method.
The ATPase activity of the intact mitochondria was inhibited
FIG. 4. Bongkrekic acid (BKA) inhibition of ATP-supported
cation uptake. Mitochondria (8.4 mg of protein) were incubated
for 8 min at 30” in a medium containing 9 mM Tris-ATP, 7.5 mM
KCl, 3.75 mM triethylamino-Pi, 1 mM MgCL, 6 X 10e8 M rotenone,
and 200 mM sucrose. Subsequent additions were 1OW M valinomy-
tin, 6 X 10-e M bongkrekic acid, 6 X 10m4 M succinate (triethyl-
amino salt), and 0.1 pg of antimycin. Initial pH of the suspension
was 7.4.
TABLE I
Inhibition of mitochondrial ATPase by bongkrekic acid
Mitochondrial protein (2 to 2.5 mg) was incubated at 30’ for 10
min in 1 ml of medium containing 6 mM ATP, 10 mM triethylamino-
Cl, 10 mM triethylamino-acetate, 15 mM KCl, 200 mM sucrose, the
ATPase-inducing agents indicated, and 20 to 25 wmoles of
bongkrekic acid per mg of protein.
Additions
Gramicidin A, 0.5 pg..
Monazomycin, 2.5 pg. .
Dinactin, 0.4 pg.. .
Monactin, 0.75 pg. . .
X-537A, 15.0 pg. . . . .
Dinitrophenol, 1CV M. . .
Dicumarol, 10-S M.
Pentachlorophenol, 10-s M. .
4,5,6,7-Tetrachloro-2-trifluoro-
methylbenzimidazole, 10-s M
1,1,3-Tricyano-2-amino-I-propene,
lo-6M...........................
Carbonyl cyanide m-chlorophenyl-
hydrazone, 5 X lo+ M..
-
-
_-
-
Pj released
Control + Bcqrekic
pvwles/mg protein X 10 ntin
1.31 0.09
1.64 0.11
1.34 0.08
1.43 0.01
0.583 0.07
1.50 0.12
1.00 0.02
1.48 0.02
0.26 0.09
0.41 0.00
1.32 0.14=
a Bongkrekic acid (10 mMmoles) per mg of protein.
by all three antibiotics. Thus, removal of the permeation
barrier appeared to remove the inhibition by bongkrekic acid.
In this way it resembles atractyloside (12, 13,48,49) and differs
from rutamycin.
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1322 Bonglcrekic Acid Inhibitim of Nucleotide Translocatim Vol. 245, No. 6
TABLE II
Failure of bongkrekic acid to inhibit ATPase in
submitochondrial particles
Intact mitochondria (1.26 mg of protein), Lubrol particles (1.32
mg of protein prepared from the same batch of whole mitochon-
dria), or sonic particles (0.44 mg) were incubated in l.O-ml volume
containing 6 mM ATP, 1.5 mM MgC12, 20 mM triethylamino-Cl, 60
mM KCI, and 75 mM sucrose for 10 min at 30”. Additions were
0.1 mM dinitrophenol, 0.05 mM bongkrekic acid, 0.1 mM atractylo-
side, and 10 pg of rutamycin.
Additions
Intact mitochondria
None.... .._.........._._.......
Dinitrophenol,
Dinitrophenol + bongkrekic acid..
Dinitrophenol + atractyloside.
Dinitrophenol + rutamycin..
Lubrol particles
0.17
1.64
0.27
0.38
0.17
None...............................
Bongkrekic acid.
Atractyloside..........................
Rutamycin.................
No MgC12..
Sonic particles
None....
Bongkrekic acid..
Atractyloside...........................
Rutamycin..............................
EDTA (5 mM)
3.21
2.97
3.28
0.40
0.42
9.12
9.10
9.10
0.71
0.59
Inhibition of Substrate Level Phosphorylation of ADP
Oligomycin (rutamycin) does not prevent the substrate level
phosphorylation of added ADP under anaerobic conditions (40))
although both atractyloside (41) and bongkrekic acid (see Fig. 3)
are potent inhibitors. Under aerobic conditions, however, the
uncoupler-accelerated oxidation of a-ketoglutarate (in the pres-
ence of Pi) is slowed by oligomycin (50), but not by atractylo-
side (23). It was found that added ADP relieved the inhibition
by oligomycin, but was not able to do so if atractyloside was
present as well as the oligomycin (23). Chappell and Greville
(50) interpreted their results as revealing an ADP requirement
for the continuation of phosphorylation and rapid respiration.
This requirement was fulfilled by the uncoupler-accelerated hy-
drolysis of the formed ATP by reversal of the oxidative phos-
phorylation enzymes. When the latter system was inhibited
by oligomycin, rapid respiration became dependent upon added
ADP (cf. Reference 51) which could be prevented from entering
the mitochondria by atractyloside (23).
The experiment of Fig. 5A illustrates comparable results with
bongkrekic acid. Dinitrophenol accelerated the ar-ketoglutarate
oxidation, presumably utilizing endogenous ADP, and this ac-
celeration was halted by rutamycin. Provision of exogenous
ADP restored rapid oxygen uptake, which could then be pre-
vented by addition of either bongkrekic acid or atractyloside.
In Fig. %, bongkrekic acid (atractyloside gave identical results)
failed to prevent the dinitrophenol acceleration of respiration,
although this was susceptible to rutamycin (Fig. 5C). The
rutamycin inhibition was relieved by added ADP (Fig. SC),
and this relief was prevented by bongkrekic acid (Fig. 50).
FIG. 5. Dinitrophenol (DNP) acceleration of a-ketoglutarate
oxidation. Each 2.5 ml of suspension contained 2.5 mg of mito-
chondrial protein, 6 mM triethylamino-ol-ketoglutarate, 20 mM
Tris-malonate, 12 mM triethylamino-Pi, 10 mM Tris-Cl, 30 mM
sucrose, 45 mM KCl, pH 7.4; the temperature was 30’; and oxygen
uptake was measured on the Gilson oxygraph. Additions were
1.2 X 1OWM dinitrophenol, 1 mM Tris-ADP, 10 fig of rutamycin,
0.02 mM bongkrekic acid (BKA), and 0.24 mM atractyloside (ATR).
In A the dashed line represents the course of reaction if the addi-
tions after dinitrophenol were omitted. In B, C, and D the
bracketed additions were made before the mitochondria (MC).
Two conclusions may be drawn from these experiments. In-
hibition by bongkrekic acid resembles that by atractyloside but
not by rutamycin, and bongkrekic acid prevents access of exoge-
nous, but not endogenous, ADP to the phosphorylation enzyme.
E$ects on Respiration Stimulated by Arsenate or Arsenate + ADP
In the absence of Pi, arsenate causes increased respiration
and uncoupling of mitochondria which is enhanced by the addi-
tion of ADP (52). The exact mechanism of these reactions is
not well understood (53), but it has been established that both
arsenate and ADP effects are prevented by oligomycin (23, 41,
52), whereas only the ilDP enhancement is susceptible to atrac-
tyloside (23, 41).
Fig. 6 shows that approximately 4 mpmoles of bongkrekic
acid per mg of protein totally prevented the ADP activation
while causing only a mild inhibition of arsenate stimulation of
respiration or NAD(P)H oxidation. However, with lower con-
centrations of arsenate and no ADP, bongkrekic acid enhanced
the arsenate-stimulated oxidation, while tending to inhibit at
higher arsenate concentrations. The effect on arsenate alone
was somewhat variable, depending upon the age of the mito-
chondrial preparation, as well as the concentration of protein,
arsenate, and bongkrekic acid present in individual experiments.
The ADP enhancement was always prevented, however. Bruni
et al. (41, 49) observed that atractyloside could enhance arsenate
effects, although this was not reported by Chappell and Crofts
(23). In our experiments, rutamycin prevented both arsenate
and ADP stimulations, as was described by the above authors.
Uptake of W-Labeled ADP and ATP by Mitochondria
Bruni, Luciani, and Contessa first described the ability of
atractyloside to prevent uptake of isotopically labeled nucleo-
tides into mitochondria (20, 49). Since that time a number of
elegant techniques have been developed for studying the kinetics
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Issue of March 25, 1970 P. J. F. Henderson and H. A. Lardy 1323
FIG. 6. Stimulation of respiration by arsenate (Asi) and ADP.
a, control; b, t-40 mpmoles of bongkrekic acid. Conditions were
similar to Fig. 2 except that Pi was omitted, glutamate-malate
substrate was replaced by 3 mM triethylamino-succinate, and the
A374-340 transmission calibration is 3’%. Additions were 6 rnM
NazHAsOa, 0.2 mM Tris-ADP, and 11 mg of mitochondrial protein.
and specificity of the nucleotide translocase and its susceptibility
to atractyloside (26, 27, 54-58).
Results obtained with bongkrekic acid by utilizing the tech-
nique described under “Experimental Procedures” are shown
in Table III. Both bongkrekic acid and atractyloside inhibited
the appearance of labeled ADP or ATP in the mitochondrial
pellet by 60 to 80%. Presumably the nonsusceptible binding
represents nucleotide contained in the compartment between
the outer and inner membranes, access to which is not con-
trolled by the translocase, as well as the nucleotide trapped in
the extramitochondrial water. Rutamycin (10 fig) did not
prevent nucleotide uptake (cf. Reference 49) and was routinely
used to prevent hydrolysis of ATP to ADP. The presence of
Pi did not alter the pattern obtained with bongkrekic acid or
rutamycin. Similar results were obtained at O”, a temperature
probably more suitable for assay of the adenine nucleotide up-
take (54,58), however, much higher concentrations of bongkrekic
acid are required to achieve a given degree of inhibition in com-
parison with experiments at 25”.
These results establish that bongkrekic acid, like atractyloside,
prevents uptake of adenine nucleotides into mitochondria.
In view of the finding that bongkrekic acid prevents transloca-
tion of ADP and ATP, it seemed unlikely that its inhibition of
oxidative phosphorylation was due to impairment of PI transloca-
tion. This has been confirmed by assaying the activity of the
Pi transport system with the swelling technique described by
Chappell and Haarhoff (59). Bongkrekic acid did not prevent
mitochondrial swelling in 100 mM (NH&.HPOa (pH 7.3) nor
the Pi-dependent swelling in (NH&.malate or (NH&.succinate
(Fig. 7), although all three activities were prevented by mersalyl,
a specific inhibitor of phosphate transport (60).
Comparison of Bongkrekic Acid with Atractyloside
So far the similarity between the effects of these two anti-
biotics has been apparent. The difference in their chemical
structures (Fig. 1 and Reference 49) led us to seek differences in
their mode of action, and these became revealed in the kinetics
of their inhibition of the oxidative phosphorylation and ATPase
systems.
By plotting the reciprocal of respiration rate against the
concentration of atractyloside, Bruni et al. (13, 61) and Vignais
TABLE III
Inhibition 0~” nucleotide uptake by mitochondria
Mitochondria (3 to 4.5 mg of protein) were incubated for 20
min in 110 mM KCl, 30 mM Tris-Cl, 1 mM Tris-EDTA, pH 7.4, with
0.3 mM (I%)-ADP or (14C)-ATP. Triethylamino-Pi (0.6 mM),
rutamycin (10 pg), bongkrekic acid, or atractyloside (50 to 60
mfimoles per mg of protein) were added as indicated. Controls
contained an equal amount of antibiotic solvent (96% ethanol),
and each value is the mean of duplicate experiments. The tem-
perature was 25”.
Nucleotide and additions Bound
nucleotide
Percentage of
control
m#mles/mg
protein
Experiment 1. (W-ADP
None............................ 8.25 100.0
Bongkrekic acid. . . . 2.50 30.3
Atractyloside...................... 3.66 44.4
Experiment 2. (l&C)-ADP
Pi. 7.21 100.0
Pi + bongkrekic acid. 1.58 22.0
Pi + rutamycin. 7.38 102.0
Pi + rutamycin + bongkrekic acid 1.65 23.9
Experiment 3. (l*C)-ATP
Rutamycin........................ 7.19 100.0
Rutamycin + bongkrekic acid. 1.30 18.1
Rutamycin + atractyloside........ 2.49 34.6
Pi + rutamycin, 7.06 98.2
Pi + rutamycin + bongkrekic acid.. 1.43 19.9
Pi
1
w
+MERSALYL
P
3
,,“Ir
iI!
?
m
CONTROL +BKA
J
TIME (MIN)
FIG. 7. Failure of bongkrekic acid (BKA) to prevent Pi-G-
tinted swelling in ammonium succinate. Light transmission
(Kodak filter No. 61 peaking at 520 rnp, light path of 2 cm) of a
suspension of 2 mg of mitochondrial protein in 5.0 ml of 100 rnM
(NH4)t-succinate and 5 mM Tris-Cl, pH 7.3, was measured after
an equilibration period of 3 min; 0.5 Mg of antimycin and 0.3 rn@-
mole of rotenone inhibited respiration, and 0.05 pmole of mersalyl
or 0.15 pmole of bongkrekic acid was present as indicated. Pi was
added to a concentration of 1.2 mM. The temperature was 30”.
and Vignais (62) obtained straight lines wit.h slopes directly
related to the concentration of ADP present. Their interpreta-
tion (cf. Reference 63) was that ADP competitively overcame
the inhibition by atractyloside. With bongkrekic acid, a
linear relationship was not obtained (Fig. 8) in the oxidative
phosphorylation assay, although ADP was still able to overcome
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1324 Bongkrekic Acid Inhibition of Nucleotide Translocatim Vol. 245, No. 6
I I I I I
0 0.5 1.0 0 0.5 1.0
myMOLES BKA/mq. PROTEIN mpM0LE.S ATR/mq PROTEIN
FIG. 8. Comparison of the efficiency of ADP in overcoming the
bongkrekic acid (BKA) (A) and atractyloside (ATR) (B) inhibi-
tions of oxidative phosphorylation. Oxygen uptake was measured
by Warburg manometry. The main compartment of each flask
contained 10 mM triethylamino-glutamate, 13.3 mM triethylamino-
Pi, 25 mM KCl, 100 mM sucrose, mitochondria (12 to 16 mg of pro-
tein), and inhibitor at the concentrations indicated. After a lo-
min thermal equilibration (30”)) oxidative phosphorylation was
initiated by the addition of ADP, 2 mg of yeast hexokinase (Sigma
type III), and glucose, to a final concentration 18 mu, from the
side arm. The oxygen uptake in 20 min was expressed as micro-
atoms of 02 per min X mg of protein (V). Each line represents a
series of flasks utilizing a single batch of mitochondria and one
ADP concentration. With atractyloside at 0.05 mM ADP the
first three points are omitted to reveal the initial sigmoidicity; this
is more clearly shown by the points included in the inset, taken
from four experiments with 0.05 mM ADP.
L I I I I
0 I 2 3
mpMOLES ANTIBIOTWmq. &OTEIi
FIG. 9. Comparison of bongkrekic acid (BKA) and atractylo-
side (ATR) as inhibitors of mitochondrial ATPase. Suspensions
of 1.0 ml containing 1.5 mg of mitochondrial protein, 6 mM Tris-
ATP, 20 mM triethylamino-Cl, 50 mM sucrose, 75 mM KCl, and
5 X 30-r M carbonyl cyanide m-chlorophenylhydrazone were
incubated for 10 min at 30”. Enzymic reactions were then stopped
by the addition of 1.0 ml of 10% trichloracetic acid, and the Pi in
each suspension was measured after centrifugation and removal
of the precipitated protein. The reaction was initiated by the
addition of mitochondria, and bongkrekic acid or atractyloside
was present at the concentrations indicated. The temperature
was 30”.
the inhibition. Fig. 8B confirms that a linear relationship is
obtained with atractyloside at 2.0 and 10.0 mM ADP under
identical conditions, although at 0.05 mu ADP and low atractylo-
side concentrations, an induction phase and sigmoidicity became
apparent (see inset, Fig. 8B). This was a reproducible result
and is also apparent in the studies of Vignais et al. (28), although
they chose to depict a linear relationship.
In Fig. 9 the effectiveness of bongkrekic acid and atractyloside
is compared with respect to their inhibition of the uncoupler-
induced ATPase. It may be seen that atractyloside was slightly
more effective at low concentrations but produced a maximal
inhibition of 350j0, whereas equal amounts of bongkrekic acid
gave 80% inhibition. Replotting these results as the reciprocal
rate against antibiotic concentration also produces a sigmoid
relationship with bongkrekic acid and a linear relationship for
atractyloside. Results t,o be presented elsewhere will show that
bongkrekic acid inhibition is uncompetitive with respect to ATP
and confirm that atractyloside inhibit,ion is competitive (62).
DISCUSSION
The experiments described in Figs. 2 and 3 confirm the report
of Welling etal. (6) that bongkrekic acid prevents both respiratory
chain and substrate level phosphorylations. The inhibition was
not due to impairment of electron transport. Rather, because
bongkrekic acid prevents the utilization of ATP in cation
transport or uncoupling, the locus of inhibition must be between
the primary high energy intermediate and external adenine
nucleotide. When the “latency” of this system was abolished
by removing the membrane barrier, bongkrekic acid no longer
inhibited although rutamycin, an established inhibitor of the
phosphorylation enzymes, was still active. Also, bongkrekic
acid prevented cu-ketoglutarate oxidation only when it was
dependent upon external, added ADP, not when the oxidation
utilized internal, endogenous ADP, and inhibited only that
portion of arsenate-accelerated respiration which is enhanced by
ADP.
It may be concluded that bongkrekic acid inhibits transloca-
tion of adenine nucleotides into mitochondria. This is con-
firmed by its ability to prevent uptake of 14C-labeled nucleotides
into mitochondria and by the close parallel of all its effects to
those of atractyloside. (Their toxicological properties are also
similar (2, 64).)
Only 1 mpmole of bongkrekic acid per mg of mitochondrial
protein was sufficient to block completely phosphorylation of
ADP, even at ADP levels of 10 IDM. Thus, at ADP concentra-
tions above about 0.1 mM, and in the absence of added Mg++,
bongkrekic acid is more effective than atractyloside. Below
this ADP concentration, atractyloside becomes equally efficacious
(see, for example, Fig. 8). A considerably higher concentra-
tion of bongkrekic acid was required to block hydrolysis of ATP
completely (about 10 mpmoles per mg of protein at 6 mM ATP),
but this was still more efficient than atractyloside. The reason
for the different susceptibilities to bongkrekic acid of the phos-
phorylation and dephosphorylation directions is under investiga-
tion. It is noteworthy that bongkrekic acid added before the
nucleotide was considerably more effective than if added at the
same time or after, and that a lag period was often observed
(e.g. Fig. 5A). It therefore seems probable that bongkrekic acid
will be as useful a tool for the study of nucleotide-requiring
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Issue of March 25, 1970 P. J. F. Hendersonand H. A. Lady 1325
mitochondrial reactions as atractyloside has been (e.g. Reference
65).
The sigmoidal curves for inhibition of oxidative phosphoryla-
tion by bongkrekic acid are extremely interesting. Since the
assay measures combined activity of the translocase and phos-
phorylation enzymes, conclusions concerning the translocase
alone must be tentative. However, it is clear that very small
changes in bongkrekic acid (or a natural effector?) concentration
change the phosphorylation system from an active to a com-
pletely inactive state, probably by inactivating the translocase, a
transition apparently opposed by increasing the level of ADP.
There is already good evidence that the translocase is an allosteric
enzyme (29), and Vignais et al. (28) have suggested that it is a
physiological control point with a steroid effector or effecters.
15. KAGAW;, Y:, AND RACKER‘, E., J. Biol. Chem.,i41’, 2461 (1966).
-^ TZAGALOFF, A., AND MCLENNAN, D. H., Biochemistry, 7, 1603
lb.
17.
18.
(1968).
The structure of bongkrekic acid more nearly resembles that
of an unsaturated fatty acid than a steroid. Significantly, it
has been reported that palmitoyl carnitine may impair availa-
bility of adenine nucleotide to mitochondria (65), and Wojtczak
and Zaluska (66) have described an inhibition of adenine nucleo-
tide translocation by oleic acid at concentrations of 25 to 50
mpmoles per mg of protein. Bongkrekic acid is therefore
considerably more potent, perhaps indicating that the enzyme
inhibitor site preferentially interacts with highly unsaturated
and acidic groupings. It is not known at the present time if
bongkrekic acid is metabolized by mitochondria, allowing the
possibility that a metabolic product is the true inhibitor.
19.
20.
21.
22.
LEE, C. P., AND ERNSTER, L., Eur. J. Biochem., 3,391 (1968).
ROBERTSON, A. M., HOLLOWAY, C. T., KNIGHT, I. G., AND
BEECHEY, R. B., Biochem. J., 108, 445 (1968).
LARDY, H. A., AND FERGUSON, S. M., Annu. Rev. Biochem., 36,
991 (1969).
BRUNI, A., LUCIANI, S., AND CONTESSA, A. R., Nature, 201,
1219 (1964).
HELDT, H. W., JACOBS, H., AND KLINGENBERG, M., Biochem.
Biophys. Res. Commun., 18. 174 (1965).
PFAFF, E., KLINGENBERG, M., AND HELDT, II. W., Biochim.
Biophys. Acta, 104, 312 (1965).
23. CHAP&L, J. B.,AND CROFTS, A. R., Biochem. J.,96,707 (1965).
24. KLINGENBERG. M.. AND PFAFF. E.. in J. M. TAGER. S. PAPA.
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26.
27.
28.
29.
30.
E. QUAGLIA&EL~O, AND E. d. SKATER (Editors), kegulation
of metabolic processes in mitochondria, Vol. 7, BBA Library,
American Elsevier Publishing Company, New York, 1966,
p. 190.
The experiment reported in Fig. 3 disclosed a phenomenon of
interest. The decrease of Pi uptake caused by bongkrekic acid
at concentrations exceeding 0.5 mpmole occurred without
appreciable decrease of cr-ketoglutarate utilization. This indi-
cates an apparent uncoupling. It is possible that when adenosine
nucleotides cannot exchange across the mitochondrial membrane
the internally synthesized ATP is used to drive the energy-linked
transhydrogenase (42) generating NADPH for glutamate
synthesis. When nucleotide movement is not blocked, the
nonenergy-linked transhydrogenation may be involved.
DUEE, E. D., AND VIGNAIS, P. V., Biochim. Biophys. Acta, 10’7,
184 (1965).
WINKLER, H. H., BYGRAVE, F.L., AND LEHNINGI(:R, A.L., J. Biol.
Chem., 243, 20 (1968).
DUEE, E. D., AND VIGNAIS, P. V., J. Biol. Chem., 244. 3932
(1969).
VIGNAIS, P. V., DUET, E. D., VIGNAIS, P. M., AND HUET, J.,
Biochim. Biophys. Acta, 118, 465 (1966).
WINKLER, H. H., AND LEHNINGER, A. L., J. Biol. Chem., 243,
3000 (1468). '
I
JOHNSON. D.. AND LARDY. H. A.. in R. W. ESTABROOK AND
31.
M. E. &L&IAN (Editors), Methods in enzymology, Fol. X,
Academic Press, New York, 1967, p. 94.
GRAVEN, S. N., LARDY, H. A., AND ESTRADA-O., S., Biochem-
istry, 6, 365 (1967).
HAGIHARA, B., Biochim. Biophys. Acta, 46, 134 (1961).
PRESSMAN, B..C., in R. W. ESTABROOK AND M. E. PULLMAN
(Editors). Methods in enzwmoloaw. VoZ. X. Academic Press.
New Yo&, 1967, p. 714. ” --’ ’
LARDY, H. A., AND WELLMAN, H., J. Biol. Chem., 201, 357
(1953).
Acknourledgments-Mrs. Doris Osthoff and Mrs. Eileen Dorsch-
ner provided excellent technical assistance, and we are indebted
to Dr. Arbianto Purwo for information on the microbial produc-
tion of bongkrekic acid. We wish to thank Professor F. M.
Strong for bringing bongkrekic acid to our attention.
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Peter J. F. Henderson and Henry A. Lardy
TRANSLOCASE OF MITOCHONDRIA
Bongkrekic Acid: AN INHIBITOR OF THE ADENINE NUCLEOTIDE
1970, 245:1319-1326.
J. Biol. Chem.
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Food Research International 21 (1994) 291-298
Fermented foods and food safety
Department ofFood Science, Agricultural University, Bomenweg 2, 6703 HD Wageningen, The Netherlands
An evaluation is presented of risk factors associated with fermented foods, in
comparison with fresh or alternatively processed foods. Cases of microbial
food-borne infection have been reported in association with fresh cheese,
sausages, fermented fish and fermented cereals. Another risk involves microbial
food intoxications due to mycotoxin contaminated raw materials, production
of bacterial toxins or possible mycotoxin production by fungal innoculants. In
addition, toxic by-products of fermentation may be produced including ethyl
carbamate and biogenic amines. From a food processing point of view, major risk
enhancing factors are the use of contaminated raw materials, lack of pasteur-
ization, and use of poorly controlled natural fermentations. Also sub-optimal
fermentation starters and inadequate storage and maturation conditions as well
as consumption without prior cooking may reduce the safety of fermented
foods. In addition to ensuring adequate processing conditions, the development
of non-toxigenic starters with ability to antogonize pathogenic microorganisms
and to degrade toxic substances needs continued attention.
Keywords: food-borne infection, intoxication, mycotoxin, ethyl carbamate, biogenic
amines, starter.
INTRODUCTION individual risk factors for the safety of the con-
sumer.
M. J. R. Nout
Food safety concerns us all as consumers, some-
times as politicians, and here particularly as food
technologists. However, consumers and food pro-
fessionals do not necessarily share the same view
concerning the major food hazards (Table 1).
This paper deals with the following questions:
(1) Are fermented foods safer than fresh or
alternatively processed foods?
(2) What risk factors can be identified in fer-
mented foods?
(3) Can fermentation principles be used to in-
crease their safety?
Like all other processed foods, fermented foods
result from a manufacturing process involving
the selection of raw materials, preparatory treat-
ments, the fermentation operation proper, preser-
vation, packaging and storage. Last but not
least, treatment of the food by the consumer in-
fluences its condition. Thus, an integral approach
should be taken when assessing the implications of
Food Research International 0963-9969/94f$07.00
0 1994 Canadian Institute of Food Science and Technology
291
FOOD INFECTION
Bacterial food-borne infections and intoxications
constitute approximately 80% of all food-related
illnesses (Waites & Arbuthnott, 1990). Food infec-
tions can occur if the following prerequisites are
fulfilled: contamination followed by survival or
growth by a pathogenic microorganism must take
place, sufficient frequency and quantity of food
must be consumed depending on the minimum
infective dose of the pathogen, and the consumer
must be susceptible to the pathogen. Particularly
the young, old, pregnant and immuno-suppressed
are more at risk than the average consumer.
Contamination
Contamination can take place during primary
production of raw materials of plant and animal
origin. In addition, it may occur during and after
processing as a result of inadequate hygiene or
packaging.

292 M. J. R.
Table 1. Food hazards: perception versus epidemiology
Source Consumersn Relative
(W importance’
(W
Microbial contamination 22 49.9
Nutritional imbalance 49.9
Environmental contaminants 48 0.05
Natural toxicants 10 0.05
Food additives 12 0.0005
Others 8
aSurvey held in The Netherlands, 1990.
’Ashwell (1990).
Survival
Most pathogenic microorganisms capable of infec-
tion are killed by pasteurisation and by exposure to
acid conditions at pH I 4-O.Other adverse environ-
mental conditions such as reduced water activity,
NaCl concentrations exceeding lo%, or chilled stor-
age are usually inadequate to prevent pathogen
survival. For instance, Listeriu monocytogenes was
shown to survive for 4 weeks at 5°C in a food sys-
tem of pH 4.18 with 13% NaCl (Cole et al., 1990).
Cases
Fermented milk products are of great economic
importance. Numerous types of cheese are pro-
duced, from pasteurised or from raw milk. The
latter procedure allows the survival of pathogens
of animal origin, e.g. Listeriu (McLauchlin et al.,
1990) and Salmonella spp. (Ratnam & March,
1986). The high buffering capacity of cheese curd
prevents a significant pH decrease during ripening,
even in the presence of active cheese starter lactic
Nout
acid bacteria. Outbreaks of salmonellosis and lis-
teriosis from raw milk cheddar and Mexican-style
cheese have been reported. In hard cheeses, con-
taminating pathogens do not survive the matur-
ation which involves several months of storage. If
the cheese milk has been pasteurised, recontami-
nation of the final product may occur. In particu-
lar, the manufacture of mould surface-ripened soft
cheeses (‘
camembert’
, ‘
brie’
, etc.) requires much
handling and is prone to re-contamination. In ad-
dition, the favourable pH caused by lactate degra-
dation by the functional fungi, e.g. Penicillium
cumemberti, enables survival and growth. It has
been estimated that 5-15% of mould ripened soft
cheeses may contain Listeriu monocytogenes due to
re-contamination (Roberts, 1990). In recent years,
outbreaks of listeriosis and Escherichiu coli gastro-
enteritis have caused much concern.
In fermented meats, pathogens, e.g. Sulmonellu
typhimurium, may survive in raw meat cured
sausages if only marginal acidification takes place
and is combined with high moisture content. For
instance, a minor outbreak of salmonellosis was
caused by fermented pork sausage. The product
had a pH of 5.7 and a, of 0.99 and was found to
contain lo6 cfu/g Sulmonellu typhimurium, among
others (Van Netten et al., 1986). Good quality
sausage should have a pH 4.5-5.0 and a, 0.92-
0.99.
The addition or in-situ production of microbial
inhibitory metabolites is considered to enhance
safety. At present, the application of bacteriocins
of lactic acid bacteria (Table 2) in food preser-
vation is limited to nisin. Nisin is applied as an
additive, or it is formed by Luctococcus Zuctis
starter cultures in the product. The genetic infor-
mation coding for nisin production has also been
Table 2. Some broad-spectrum bacteriocins of lactic acid bacteria
Bacteriocin Produced by Activity Heat Approved Applied Reference
against stability
Nisin-A Lmtococcus lactis Gram + 10 min, GRAS Yes Delves-Broughton
100°C (1990)
Bulgarican Lmtobacillus delbriicki Gram + Abdel-Bar & Harris
ssp. bulgaricus and - (1984)
Pediocin A Pediococcus pentosaceus Gram + 1 h, 100°C Daeschel &
and - Klaenhammer (1985)
Pediocin AcH P. acidilactici (H) Gram + Bhunia et al. (1988)
and -
Reuterin Lb. reuteri Gram + FDA Daeschel(l989)
(non peptide) and -, fungi

Fermentedfoods andfood safety 293
cloned and expressed in different cheese starter
bacteria (Coghlan, 1990), but these are not as yet
applied commercially. Nisin is produced by L. luctis
and has a rather broad-spectrum antimicrobial
action including many Gram-positive bacterial
species. It is able to inhibit spore germination of
Clostridium botuliraum in canned foods and kill
Listeria spp. in raw (fermented) food. In order to be
attractive as food preservatives, bacteriocins must
have a broad-spectrum of antimicrobial activity,
preferably including fungi. In addition, they must
be stable to heat and other adverse conditions.
Obviously, only non-toxic and non-allergenic pro-
ducts of GRAS-organisms may be considered for
application.
In raw fish and fermented raw fish, Vibrio para-
haemolyticus will survive quite well. In Japan, in
particular, K parahaemolyticus infections are com-
mon and can be associated directly with the
custom of consuming raw (fermented) fish. Lack
of heating or smoking is supposed to be the
reason why fermented fish (salmon, halibut, her-
ring) was much more frequently (25% of n = 89
samples) contaminated with Listeriu monocyto-
genes than hot-smoked (9% of n = 496) or cold-
smoked (14% of n = 324) fish (Jemmi, 1990).
In many tropical countries, raw cereals and pulses
are allowed to undergo uncontrolled natural fer-
mentations in order to enhance their flavour and
digestibility. No salt is present in these high-mois-
ture products. As a result of the activity of Entero-
bacteriaceae and lactic acid bacteria, a moderate
extent of acidification occurs with pH values rang-
ing from pH 4.5-5.5. These environmental con-
ditions enable the survival of pathogenic bacteria.
Normally this type of product requires cooking
prior to consumption. Thus, if consumed immedi-
ately after cooking, one would not expect any risk
of food infection. In practice, however, the food
utilization habits of the consumer appear to play
a crucial role. In particular, these naturally fer-
mented cereal- or cereal/legume-based porridges
are used as weaning foods in the tropics. Due to
time constraints there is a tendency to prepare in
advance and feed left-overs and there is a wealth
of literature demonstrating the poor hygienic
conditions of such traditional weaning foods. It
would appear obvious that the daily intake of
heavily contaminated food causes a significant
incidence of food-borne infection symptoms in-
cluding diarrhoea. However, there is very little
evidence for a direct correlation between incidence
of infectious diarrhoea and faecal contamination
moisten raw material
iL overnight II3
/ -s7
naturally fe’
hented accelerated acidification
1 further processing 1
Fig. 1. Principle of accelerated acidification by ‘
back-slopping’
.
of weaning food (Lloyd-Evans et al., 1984). This
can be due to several reasons. Firstly, viruses
(Enterovirus, Rotavirus) also play an important
role in the etiology of diarrhoea. Virus control
requires proper cooking of food and water; no
untreated water must be used to ‘
dilute’ boiled
porridges. Secondly, the acidity of the traditional
products may be inadequate to kill the contami-
nating pathogens.
It has been shown that under simple processing
conditions, the rate and extent of acidification of
natural fermentations can be improved signifi-
cantly by enrichment of inocula using the ‘
back-
slopping’ method (Fig. 1). This approach was
successful with a variety of root crops, cereals and
legumes and had significant bactericidal effect
in challenge tests with a variety of Enterobac-
teriaceae (Nout et al., 1989). In acidifying maize
of pH 4.144 faecal coliforms died at a rate of
approximately 1 log cycle per hour.
The fermentative preservation of animal feed by
ensiling is of importance. Fresh grasses, fodder
crops or industrial by-products, e.g. sugar beet pulp,
are packed or heaped while creating anaerobic
conditions to stimulate the activity of lactic acid
bacteria and fermentative yeasts which will de-
crease the pH and compete with less desirable
microorganisms. A regularly occurring problem is
that of fungal spoilage of silage, notably with
Penicillium roqueforti and Aspergillus fumigatus. It
was demonstrated that the increased pH in silage
spoiled by P. roqueforti enabled the survival of
pathogens including Listeriu spp. In turn, this
could contribute to the maintenance of contami-
nation cycles of pathogenic microorganisms.
FOOD INTOXICATION
Food intoxications, either acute or chronic, may
occur depending on the quantity and nature of

294 M, J. R. Nout
the ingested toxin. Consumer sensitivity towards
toxins may vary considerably with general state of
nutrition and health, and with the dietary pattern.
In the context of this paper, three sources of
toxins will be discussed: those already present in
the raw material, microbial toxins produced dur-
ing or after processing, and toxic by-products of
fermentation.
Raw materials
A number of raw materials naturally contain toxic
substances, for instance cyanogenic glycosides
(Reddy & Pierson). In addition, environmental
contaminants such as pesticides, herbicides and
hormones may be present. There is little evidence
that food fermentation has a diminishing effect on
such residues. In the field and during storage,
plant foods, in particular, may become contami-
nated with mycotoxins. The fate of aflatoxins dur-
ing food fermentation has been studied by several
investigators.
Aflrtoxin B t Aflatoxin B,,
Afirtoxicol A Anatoxicol B
Fig. 2. Detoxification of aflatoxin B,.
0 0
2 O I
I c
0 0 -3
Aflatoxin B,
Fig. 3. Detoxification of aflatoxin B, by opening of the
lactone ring.
Groundnut presscake or maize used as a raw
material for the production of fermented prod-
ucts, e.g. Indonesian oncom and Ghanaian kenkey,
may be contaminated with aflatoxins. Also, animal
feed ingredients may have considerable mycotoxin
levels. The fate of aflatoxin B, during food fer-
mentation has been investigated in a variety of
products. Fungi involved in food fermentations,
for instance Rhizopus oryzae (= R. arrhizus) and
R. oligosporus are able to reduce the cyclo-
pentanon moiety which results in aflatoxicol A
(Fig. 2). This appears to be a reversible reaction.
Under suitable growth medium conditions (e.g.
presence of organic acids), aflatoxicol A is irre-
versibly converted into its stereo-isomer aflatoxi-
co1 B (Nakazato et al., 1990). Aflatoxicol is
approximately 18 times less toxic than aflatoxin
B,.
In lactic fermentations at pH I 4.0, aflatoxin B,
is readily converted into aflatoxin B,, (Fig. 2)
which is also less toxic. Both biotransformations
thus reduce the toxicity but there is no complete
detoxification unless the lactone ring of the
aflatoxin molecule is broken (Fig. 3). This would
correspond to loss of fluorescence at 366 nm. It
was found that such loss of fluorescence correlates
with reduced mutagenicity. Screening fungi for
the ability to reduce fluorescence in aflatoxin B,
medium revealed that certain Rhizopus spp. were
able to degrade 87% of aflatoxin B, into non-
fluorescent substances, of as yet unknown nature
and toxicity (Bol & Smith, 1989). This might pro-
vide opportunities for detoxification of food and
feed in solid substrate fungal fermentations.
Microbial toxins
Microbial toxins may be produced by contaminat-
ing microorganisms. In some cases, the functional
flora has been found to be toxigenic.
Contaminants
In large pieces of meat, e.g. country cured ham or
in insufficiently heated or cured sausages, there is
a realistic chance that Clostridium botulinurn or
Clostridium perfringens could grow and produce
toxins, if brining and drying are inadequate. It is
therefore essential to ensure an adequate combi-
nation of inhibitory factors (NaCl, nitrite, water
activity, pH) or to apply heat treatments to avoid
clostridium poisoning.
In cheese made from raw milk, Staphylococcus
aureus may grow and produce enterotoxins. As

S. aureus is inhibited in the presence of competing
microflora, the presence of actively growing starter
cultures strongly reduces the chance of entero-
toxin formation.
Tempe technology plays an important role in
providing high quality protein from plant origin,
especially in south-east Asia. Most commercial
tempe makers use soybeans as a raw material. It
has been shown that the acidification taking place
during the preparatory soaking of the soybeans,
plays a role in the development of the tempe
microflora. In particular, poorly acidified beans
allowed the survival and growth of pathogenic
and toxinogenic bacteria including Bacillus cereus,
Yersinia enterocolitica and S. aureus. It has been
shown that growth of Salmonella spp., Entero-
bacteriaceae and S. aureus during the stage of
fungal fermentation is inhibited if competing
lactobacilli are present; if S. aureus does grow it is
unable to produce significant amounts of entero-
toxin (Nout & Rombouts, 1990). Moreover,
staphylococcal enterotoxins are not very heat re-
sistant when present in tempe, and since tempe
must be cooked or fried before consumption, the
risks of food-borne infection or intoxication are
Bon$trck acid
small indeed. Soybean tempe has never been
incriminated as a cause of food-borne disease.
However, tempe ‘
bongkrek’
, made from coconut
presscake in Central Java, Indonesia, may enable
the multiplication of Pseudomonas cocovenenans
which produces the toxins bongkrek acid and
toxoflavin (Fig. 4). Tempe ‘
bongkrek’ has caused
several fatal poisonings. Interestingly, Ko (1985)
established that a large Rhizopus oligosporus inocu-
lum size, or incorporation of 2% NaCl are ade-
quate to prevent the growth and toxin production
of P. cocovenenans in this type of tempe.
In ensiled animal feed, fungal spoilage by Peni-
cillium roqueforti and Aspergillus fumigatus is
common. All of 34 P. roqueforti strains isolated
produced P.R.-toxin, and 6 of 13 isolates of A. fwni-
gatus produced fumitremorgens, verruculogen and
TR2-toxin, in laboratory media (Gedek et al.,
1981). In practice however, there is little evidence
of accumulation of such toxins in silage or of
poisoning of cattle (Nout et al., 1993).
In fermentations involving non-cooked raw
materials, the combined effect of water activity,
salt concentration, acidity, anaerobiosis, tempera-
ture and microbial competition must be optimized.
Toxoflwin
H3C-N
I I I
o-c
N/CN/N
I
c% C7H7NSo2
Fig. 4. Toxins of Pseudomonas
cocovenenans.

296 hf. J. R. Nout
In this respect, predictive modelling of the be-
haviour of toxinogenic microorganisms such as
CZostridium botulinurn (Lund et al., 1990) is a use-
ful tool. In addition, the use of competitive fer-
mentation starters has been successfully used to
suppress the multiplication of Salmonella spp.,
Listeria monocytogenes and Escherichia coli in
meat and cheese model systems (Earnshaw et al.,
1989).
CH3
- CH,- O-C -NH2

0
Fig. 5. Ethyl carbamate (urethane).
Table 3. Occurrence of ethyl carbamate io fermented foods”
FunctionaZjZora
Most starter organisms used in commercial prac-
tice are considered to be non-pathogenic. Excep-
tions may be Staphylococcus saprophyticus and
S. xylosus which are part of certain meat curing
inocula (Hammes, 1988). As the latter organisms
have also been isolated from human infections,
their pathogenicity merits further study.
The toxinogenicity of fungal starters, how-
ever, has recently been of concern. In particular,
PeniciZZium spp. used in cheese (P. roqueforti,
P. camembertii) and in meat (P. chrysogenum,
P. nalgiovense) are, in principle, able to produce
mycotoxins. P. roqueforti may produce roque-
fortine C and A, and mycophenolic acid in test
media. P. camembertii produces some cyclo-
piazonic acid in the rind of camembert cheese if
this is stored without refrigeration. At present,
non-toxinogenic strains of these fungi are not
known and a project is underway to obtain non-
toxinogenic mutant strains (Leistner, 1990).
Product Number of Average level
samples (ppb)
Cheese 16 NDb
Tea 6 ND
Yoghurt 12 0.4
Cider 8 0.6
Bread 30 1.7
Malt beverages 69 1.8
Bread, toasted 9 52
Soy sauce 12 18
Wine 6 18
Sake 11 52
‘
Literature data.
bND = Not detectable.
In cured meat, fungal starters contribute to the
aroma, the quality of the skin, and product safety
by suppressing wild strains and their metabolites.
Non-toxinogenic strains of P. nalgiovense and
P. chrysogenum (white mutant) are marketed as
meat curing starters. More than 50% of P. nalgio-
vense and P. chrysogenum isolated from fermented
meats are toxinogenic when tested on laboratory
media (Leistner & Eckardt, 1979). Although very
little information exists on the production and
chemical stability of mycotoxins in complex food
systems such as meat, some national food laws
require that no toxinogenic fungi should be cul-
tivable from fungal fermented products. Surely,
this is no guarantee that mycotoxins are absent!
results from the esterification of ethanol with
carbamic acid (Canas et al., 1989). The latter
can be formed from several precursors including
naturally occurring citrulline, as well as yeast
metabolites from L-Arginine and L-Asparagine,
e.g. urea and carbamylphosphate. In addition,
vicinal diketones, and HCN liberated from cyano-
genie glycosides act as precursors. Heat and light
enhance the formation of ethyl carbamate. Table 3
summarises literature data on its occurrence in
foods and beverages (Hasegawa et al., 1990). In
most countries there is no legislative limit, but a
level of 10 ppb was suggested by FAO/WHO for
soft drinks, and 30400 ppb was suggested for
various alcoholic beverages by the Canadian Gov-
ernment.
The mechanism of ethyl carbamate formation is
poorly understood. Research with wine and stone
fruit (cherry, plum) fermentations indicate that re-
ducing the level of precursors by enzyme treat-
ment, selection of yeast strains and control of
fermentation conditions, and treatment of the
pH adjusted fermented pulp with CuSO, could
be useful in keeping the ethyl carbamate levels to
a minimum.
By-products of fermentation Biogenic amines
Ethyl carbamate
A substance occurring in a variety of fermented
foods is ethyl carbamate (urethane) (Fig. 5), a
carcinogenic and mutagenic compound which
Biogenic amines are a group of mildly toxic com-
pounds which can be formed in fermented foods,
mainly by decarboxylation of amino acids (Table 4).
Approximately 1000 ppm is supposed to elicit
toxicity. From a ‘
good manufacturing practice’
Range
(ppb)
ND-4
ND-4
ND-8
ND-13
2-14
ND-84
7-40
3-116

Table 4. Major biogenic amines
Biogenic amine Formula Precursor
Ethylamine
CzH,N
CH,CH2NH,
Putrescine
C,H,2N2
H,N(CHz),NHz
Histamine
CsHgN,
CH,CH2NH,
Cadaverine
GH,,N2
H,N(CH&NHz
Tyramine
CsH, @N
Phenylethylamine
CaH,,N
Tryptamine
C,OH,,NZ
CH,CH,NH,
Ala
0l-n
His
LYs
Tyr
Phe
Try
H
point of view, levels of 5&100 ppm histamine,
100-800 ppm tyramine and 30 ppm phenylethyl-
amine, or a total o:f 100-200 ppm are regarded as
acceptable. Biogenic amines are especially associ-
ated with lactic fermented products, particularly
wine, cheese, fish and meat and very low levels
also occur in fermented vegetables (Fig. 6). The
major biogenic amine producers in foods are
Enterobacteriaceae and Enterococci. Most func-
tional lactic acid bacteria do not produce signifi-
cant levels of biogenic amines. Presence of free
amino acids, low pH of the product, high NaCl
concentrations, and microbial decarboxylase
safe
i5Gl
1OOppm
accepted
1OOOppm
hazardous
>2000ppm
ouda cheese
Ibriekamembertl
blue cheese/gorgonzolal
lterasi (fish paste)/
Fig. 6. Biological amines in fermented foods (literature data:
sum of concentrations of individual amines in ppm).
activity correlate with higher levels of biogenic
amines (Ten Brink et al., 1988). In meat products,
species of Enterobacteriaceae were associated with
cadaverine, and lactobacilli with tyramine for-
mation. Also sauerkraut may contain varying
levels of biogenic amines, due to the large vari-
ations in the naturally selected microflora. In cheese,
Enterobacteriaceae, heterofermentative lactobacilli
and Enterococcus faecalis were associated with
considerable production up to 600 ppm of bio-
genie amines including phenylethylamine.
Pasteurisation of cheese milk, hygienic practice
and selection of starters with low decarboxylase
activity are measures to avoid
these undesirable products.
CONCLUDING REMARKS
the accumulation of
Due to the competitive activity and the meta-
bolites of starter microorganisms, many fermented
foods are a less likely vehicle for food infection or
intoxication than fresh foods. On the other hand,
they are often not as stable as canned or frozen
foods, and good hygienic practice during their
manufacture strongly contributes to their dura-
bility and safety. The following risk factors are of
importance:
(4
(W
(cl
Cd)
(e>
the use of previously contaminated raw ma-
terials;
lack of pasteurisation;
the use of poorly controlled natural fermen-
tations, or of sub-optimum fermentation
starter cultures;
inadequate storage or maturation condi-
tions enabling survival of pathogens, or
growth and toxin production;
consumption without prior heating.
How can these risks be minimized? Obviously, it
is essential to ensure the wholesomeness of raw
materials. Food fermentation cannot be used as a
tool to produce first quality products from second
quality raw materials.
In addition, further optimisation of starter cul-
tures either by conventional selection and muta-
tion, or by recombinant-DNA manipulations can
result in increased levels of safety of fermented
foods. In particular, selection of starters which are
not toxinogenic, which antagonize pathogenic
microorganisms, which produce broad-spectrum
bacteriocins, or which have detoxifying ability
should have priority.

298 A4. J. R. Nout
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Bo 1996 Stockton Press All rights reserved 0007-1188/96 $12.00 M
Enhanced vasocontraction of rat tail arteries by toxoflavin
*.tZunzhe Wang, tMeisheng Ma & l,*Rui Wang
*Departement de physiologie, Universite de Montreal, C.P. 6128, Succ. Center-ville, Montreal, Quebec, Canada H3C 3J7 and
tDepartment of Parasitology and Microbiology, Weifang Medical College, Weifang, P.R. China
1 It has been suggested that the toxic effect of toxoflavin (TXF) produced by Pseudomonas cocovenenas
is mainly due to the impairment of electron transfer of the mitochondrial respiratory chain. However,
the cardiovascular effect of TXF is unknown. In the present study, the effect of TXF on the isometric
contraction of rat isolated tail artery strips and the underlying mechanisms were investigated.
2 The basal force of the tissues was not affected by the toxin. However, the application of TXF before
or during KCl (60 mM) stimulation potentiated KCl-induced vasocontraction, specifically the tonic phase
of the contraction.
3 When the vessel strips were precontracted with phenylephrine (Phe), TXF further enhanced the tonic
contraction of the tissue. Pretreatment of tissues with TXF also potentiated subsequent vasocontraction
induced by Phe. The vasocontractor effects of TXF and Phe, however, were not additive.
4 The vascular effect of TXF was not mediated by oxygen-derived free radicals since catalase and SOD
did not affect TXF-enhanced vasocontraction. In contrast, the vasocontractor effect of TXF was
dependent on extracellular Ca2+ and abolished by nifedipine (a Ca2+ antagonist). TXF also had no
effect on caffeine- or U46619-induced vasocontraction.
5 It is suggested that TXF may potentially contract blood vessels via its effect on Ca2+ channels. This
effect of TXF depends on the contractile status of the vascular tissues.
Keywords: Toxoflavin; vascular smooth muscle; contraction; phenylephrine; calcium channel; oxygen-derived free radicals
Introduction
Consumption of fermented corn meal, banana, and coconut
often causes food poisoning due to contamination with Pseu-
domonas cocovenenas (Zhao et al., 1990). Toxoflavin (TXF)
and bongkrekic acid are two toxins produced by Pseudomonas
cocovenenas and responsible for the mortality and morbidity of
Pseudomonas cocovenenas poisoning (van Damme et al., 1960).
TXF, known as xanthothricin, can also be produced by a
culture of the genus Streptomyces (Machlowitz et al., 1954).
That the same toxin has two different biological sources sig-
nifies a biological function of TXF. It has been suggested that
the toxic effect of TXF may result from the formation of hy-
drogen peroxide (Latuasan & Berends, 1961). Particularly,
TXF may act on iron-sulphur cluster in NADH-Q oxidor-
eductase, participating or promoting the Fenton reaction
(Koppenol, 1993; Giulivi et al., 1995) to generate H202 (Xu et
al., 1990). Due to the lack of knowledge on the spectrum of
biological effects ofTXF as well as the relative mechanisms, no
effective detoxicating method is yet available for TXF poi-
soning.
An increased generation of oxygen-derived free radicals
(OFR) and a simultaneously decreased production of anti-
oxidants, such as superoxide dismutase (SOD) and vitamin E,
have been suggested as being involved in human essential hy-
pertension (Kumar & Das, 1993). OFR may act directly on
vascular endothelium (Wu et al., 1992) and smooth muscle
cells (SMC). In the former case, OFR may inactivate en-
dothelium-generated nitric oxide (NO) (Ikeda et al., 1994) or
impair the production of NO (Seccombe et al., 1994). In the
latter case, it has been shown that hydroxyl radicals generated
by metal ions plus hydrogen peroxide contracted single SMC
isolated from the basilar artery of the rat (Steele et al., 1991).
Assuming that the biological effects of TXF were due to the
generation of OFR, TXF should be able to induce vasocon-
traction and to increase blood pressure. However, the vascular
effect of TXF and the underlying mechanisms are unknown to
Author for correspondence.
date. In the present study, the vascular effect of TXF was
studied in rat isolated tail artery strips, precontracted with
either KCl or phenylephrine. It was found that TXF enhanced
the vasocontraction in an extracellular calcium-dependent
manner, irrespective of the production of oxygen-derived free
radicals.
Methods
Measurement of tension development of rat tail artery
strips
Tail arteries were isolated from male Sprague-Dawley (SD)
rats (150-200 g) (6-8 weeks old). Segments of tail artery of
approximately 1.5 cm in length were cut into helical strips. The
strips were then mounted in a 10 ml organ bath chamber filled
with a Krebs bicarbonate saline (bubbled with 95%02/
5%CO2) and were mechanically stretched to achieve a basal
force of approximately 0.7 g. Tissues were routinely allowed to
equilibrate for 1 h before the start of experiments. In-
domethacin (1 yLM) was routinely added to the Krebs saline
which was composed of (in mM): NaCl 115, KCl 5.4, MgSO4
1.2, NaH2PO4 1.2, NaHCO3 25, glucose 11, and CaCl2 1.8. The
endothelium was removed by a rubbing procedure and the lack
of endothelium was confirmed by the failure of acetylcholine
(1 jgM) to relax the tissue. The tension development was mea-
sured with an FT 03 force displacement transducer (Grass Ins.
Co., Quincy). Data acquisition and analysis were accomplished
using a Biopac system (Biopac Systems, Inc., Golata), in-
cluding the MP 100 WS acquisition units, TCI 100 amplifiers,
an AcqKnowledge software (3.01), universal modules and a
Macintosh computer.
Chemicals and data analysis
TXF was prepared in Weifang Medical College, P.R. China,
according to the method of van Damme et al. (1960). Briefly,
strains of Pseudomonas cocovenenas (T7707-b) were cultured at
British Journal of Pharmacology (1996) 117, 293 298

Z. Wang et al Vascular effect of toxoflavin
280C under continuous rotation (180 turns min-') for 48 h
(Zhao et al., 1990). Consequently, the culture medium was
saturated with (NH4)2SO4 to remove bacterial and proteins.
TXF was repeatedly extracted from the filtered medium with
chloroform and petroleum ether. The content and the purity of
TXF were determined with two ultraviolet spectrophotometers
(Model 751 and Model UV-210A), respectively. Figure 1
shows the chemical structure of TXF. The present study was
performed with two batches of TXF of which the stock con-
centrations were 362 ,ug ml-' and 500 jug ml-', respectively.
Phenylephrine (Phe), acetylcholine, caffeine, U-46619 (9,11-
dideoxy-1 la, 9La-epoxymethano-prostaglandin F20), indo-
methacin, and other chemicals were purchased from Sigma.
Data are expressed as X+s.e. unless otherwise specified.
Student's t test or analysis of variance in conjunction with the
Newman-Keul's test were used where applicable. Group dif-
ferences were considered statistically significant at the level of
P< 0.05.
Results
Effect of TXF on KCl-induced vasocontraction
KCl (60 mM) induced a biphasic contraction of vascular strips
isolated from rat tail artery. In the presence of TXF
(3 jug ml-'), the phasic contraction (peak contraction) induced
by KCl did not change whereas the tonic contraction was
enhanced (left panel of Figure 2a). The total contraction ofthe
tissue within 10 min of the application of stimuli was calcu-
lated as the integrated contraction, shown as the shaded area
under the contraction curve in Figure 2a. KCl-induced in-
tegrated contraction was greater (14.04 + 1.80 g.min) in the
presence of TXF than in the absence of TXF
(11.15+1.52 g.min, n=7) (P<0.05). TXF (0.3-ljIg ml-')
alone had no effect on the basal force of rat tail artery strips.
The basal force was 0.68 +0.06 g and 0.68 +0.06 g before and
10 min after addition of TXF 1 ,ug ml-', respectively (n = 12,
P> 0.05). Even 30 min after the application of TXF, the basal
force still remained unchanged (data not shown). To determine
whether the vasocontractor effect of TXF depended on the
contractile status of the tissue, TXF was applied to the tissue
immediately after the KC1-induced contraction reached its
peak. The right panel of Figure 2a shows that, in the presence
of KCl, TXF (3 pig ml-') further enhanced the tonic con-
traction of the tissue. In this group of experiments, KCl-in-
duced integrated contraction was greater (15.62+ 1.43 g.min)
with the addition of TXF than that without TXF
(9.79+ 1.04 g.min, n=8) (P<0.01). TXF was also applied to
the tissue at the middle of the tonic contraction induced by
KCl (Figure 3). In this case, TXF (3 ,ug ml-') still effectively
enhanced the tonic contraction (n = 3).
Effect of TXF on phenylephrine-induced vasocontraction
In the presence of TXF, phenylephrine (Phe)-induced con-
traction of tail artery strips was significantly enhanced (Figure
4). The concentration-dependent contraction curve of tissues
to PHE was shifted to the left after the tissues were pretreated
with TXF (1 ,ug ml-') (Figure 5). In the next group of ex-
periments, the tissue contraction was firstly induced by Phe at
a fixed concentration (0.3 or 1 ,UM). When the contraction of
the tissues reached the plateau phase, TXF was added at ac-
cumulated concentrations. At a concentration of0.01 gig ml-',
TXF did not modify Phe-induced vasocontraction. When the
concentration of TXF was further increased, Phe-induced va-
socontraction was significantly potentiated (Figure 6). How-
ever, when the concentration of TXF was higher than
10 yg ml-', vasorelaxation occurred irreversibly, possibly due
to the tissue poisoning (data not shown). To test further
whether the effects of TXF and Phe were additive, TXF at a
single concentration (1 jug ml-') was added to the bath when
Phe (0.3 to 30 kLM)-induced vasocontraction reached the pla-
H3CNr--- clvNll
NI N
CH
Figure 1 The chemical structure of toxoflavin.
a
TXF
5 0.1
9
KCI 60 mM
b
_
_
To
0
4-1
cJ 120
00
0i
a)
4-
~0
*
T
Figure 2 Effect of TXF on rat isolated tail artery strips
precontracted with KCl (60mM). Pretreatment of the tissues with
TXF (1 ygm1-') enhanced KCl-induced integrated contraction,
shown at the left of panel (a) and columns at left of (b) (n = 7).
Application of TXF following KCl stimulation, shown at the right of
panel (a) and columns at right of (b) (n = 8), also significantly
enhanced KCl-induced integrated contraction. In (b) TXF enhanced
KCl-induced integrated contraction was compared with that in the
absence of TXF. Open column: 60mm KCl; solid columns: 60mM
KCl plus TXF 1 Ygml-
m . Shaded areas in (a) indicate the calculated
area as the integrated contraction. KCl-induced integrated contrac-
tion in the absence of TXF was taken as the control (100%).
*P<0.01; **P<0.05.
teau phase as shown in Figure 7a. Irrespective of the con-
centrations of Phe, TXF always induced a similar amount of
enhancement of isometric contraction of the tail artery strips
(Figure 7b).
Extracellular calcium-dependency of the vasocontracting
effect of TXF
Without calcium in the bath solution, Phe (1 jM) had no effect
on the isometric tension of tail artery strips (Figure 8a). In the
presence of Phe, the stepwise addition of calcium to the bath
solution induced a graded vasocontraction maximally devel-
oped around 1 mm calcium. Similarly, Phe and TXF together
in the absence of extracellular calcium did not affect the iso-
metric tension of tail artery strips. However, the extracellular
calcium-dependent Phe-induced contraction of the tissues was
significantly enhanced in the presence of TXF (Figure 8b).
Furthermore, the extracellular calcium-dependent and Phe/
TXF-induced contraction of the tissues was inhibited by nife-
dipine (1 jM), a classical calcium channel blocker. One ex-
ample of the effect of nifedipine is shown in Figure 8a.
294
5 min 0.1 g
TXF

Makalah Tempe Bongkrek : Asam Bongkrek dan Toksoflavin)

Makalah Tempe Bongkrek : Asam Bongkrek dan Toksoflavin)

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