Dissertation (PhD)--University of Stellenbosch, 2007. / ENGLISH ABSTRACT: Aflatoxins are mycotoxins predominantly produced by the filamentous fungi Aspergillus
flavus and Aspergillus parasiticus. Aflatoxin B1 (AFB1), the most abundant aflatoxin, is
highly mutagenic, toxic, carcinogenic and teratogenic to humans and animals and is
particularly correlated with the incidence of hepatocellular carcinoma in parts of Africa,
China and South East Asia. In this regard aflatoxin is classified as a type I human
carcinogen by the International Agency for Research on Cancer. Furthermore, aflatoxin
contamination of food and feed is responsible for extensive economic losses due to loss
of crops and farm animals.
In spite of regulations regarding acceptable levels of aflatoxin in food, aflatoxin
contamination remains a serious worldwide problem, especially in developing countries
where it occurs predominantly in dietary staples. Inactivation of aflatoxin by physical
and chemical methods has not yet proved to be effective and economic. However,
biological detoxification offers an attractive alternative for eliminating toxins as well as
safe-guarding the desired quality of food and feed.
In this study, the biological degradation of AFB1 by bacteria and fungi was
investigated. Several bacteria, including Rhodococcus spp., as well as white rot fungi
have the potential to degrade a wide range of polycyclic hydrocarbon compounds due to
the large repertoire of enzymes they produce and therefore the ability of some of these
microorganisms to degrade AFB1 was investigated. Effective degradation of AFB1 by
intracellular extracts of Mycobacterium fluoranthenivorans sp. nov. DSM 44556T,
Nocardia corynebacterioides DSM 20151 and N. corynebacterioides DSM 12676 was
demonstrated. Furthermore, AFB1 was effectively degraded by liquid cultures as well as
intra- and extracellular extracts of Rhodococcus erythropolis DSM 14303. Significant
(P<0.001) reduction in AFB1 was observed following treatment with R. erythropolis
extracellular extracts with only 33.20% residual AFB1 after 72 h. Results indicated that
the degradation by R. erythropolis DSM 14303 is enzymatic and that the enzymes are
constitutively produced. The degradation of AFB1 when treated with R. erythropolis
DSM 14303 extracellular extract coincided with a total loss of mutagenicity. In addition,
treatment of AFB1 with culture fractions containing recombinant 2,3-dihydroxybiphenyl dioxygenase, which was produced through extracellular expression of the bphC1 gene of
R. erythropolis DSM 14303 in Escherichia coli BL21, resulted in significant (P<0.0001)
degradation (49.32%) and reduced mutagenic potency (42.47%) of the molecule.
Significant (P<0.0001-0.05) degradation of AFB1 was obtained following treatment
with culture extracts containing laccase enzyme produced by white rot fungi (17.10-
76.00%), purified fungal laccase from Trametes versicolor (1 U/ml, 87.34%) as well as
with recombinant laccase produced by Aspergillus niger (118 U/L, 55.00%).
Furthermore, treatment of AFB1 with purified fungal laccase enzyme (1 U/ml) resulted in
loss of the mutagenic potency of the molecule. The decrease in the fluorescence and
mutagenic properties of AFB1 following treatment with the microbial preparations imply
changes to the furofuran- and/or lactone rings of the molecule.
The current study contributes towards developing genetic engineered microbial
strains which could be applied as an important bio-control measure. Such strains could
exhibit multifunctional technological properties including degradation of AFB1, to
significantly improve the quality, safety and acceptability of food. / AFRIKAANSE OPSOMMING: Aflatoksiene is mikotoksiene wat hoofsaaklik deur die filamentagtige fungi, Aspergillus
flavus en Aspergillus parasiticus geproduseer word. Die algemeenste aflatoksien,
aflatoksien B1 (AFB1), is hoogs mutagenies, toksies, karsinogenies en teratogenies vir
mense en diere. Veral in sekere dele van Afrika, China en Suid-Oos Asië bestaan daar `n
korrelasie tussen aflatoksien en die voorkoms van hepatosellulêre karsinoom en gevolglik
word aflatoksiene as `n tipe I menslike karsinogeen deur die Internasionale Agentskap vir
Kankernavorsing geklassifiseer. Aflatoksien kontaminasie in voedsel het ook `n
ekonomiese impak as gevolg van verlies aan landbougewasse en diere.
Ten spyte van maatreëls betreffende die toelaatbare vlakke van aflatoksiene in
voedel, is aflatoksien kontaminasie steeds `n groot probleem wêreldwyd, veral in
ontwikkelende lande waar dit hoofsaaklik in stapelvoedsel voorkom. Huidiglik is die
inaktivering van aflatoksiene deur fisiese en chemiese metodes nie effektief en
ekonomies nie. Daarteenoor bied biologiese tegnieke `n gunstige opsie vir die
eliminering van die toksiene, terwyl die organoleptiese eienskappe van die voedsel steeds
behoue bly.
Hierdie studie fokus op die biologiese afbraak van AFB1 deur bakterieë en fungi.
Verskeie bakterieë, insluitend Rhodococcus spp., sowel as witvrot fungi produseer `n
verskeidenheid ensieme wat hulle in staat stel om `n wye reeks polisikliese
hidrokoolstofverbindings af te breek en gevolglik is afbraak van AFB1 deur sommige van
hierdie mikroörganismes bestudeer. Effektiewe afbraak van AFB1 deur intrasellulêre
ekstrakte van Mycobacterium fluoranthenivorans sp. nov. DSM 44556T, Nocardia
corynebacterioides DSM 20151 en N. corynebacterioides DSM 12676 is aangetoon.
AFB1 is ook effektief in vloeibare kulture sowel as intra- en ekstrasellulêre ekstrakte van
Rhodococcus erythropolis DSM 14303 afgebreek. `n Beduidende (P<0.001) afbraak van
AFB1 is waargeneem na behandeling met R. erythropolis DSM 14303 ekstrasellulêre
ekstrakte, met slegs 33.20% oorblywende AFB1 na 72 h. Resultate het getoon dat die
afbraak deur R. erythropolis DSM 14303 ensimaties is en dat die ensieme konstitutief
geproduseer word. Afbraak van AFB1 deur R. erythropolis DSM 14303 het ook tot `n
totale verlies aan mutagenisiteit gelei. Verder het behandeling van AFB1 met rekombinante 2,3-dihidroksiebifenieldioksiginase fraksies wat geproduseer is deur
ekstrasellulêre uitdrukking van die bphC1 geen van R. erythropolis DSM 14303 in
Escherichia coli BL21, beduidende (P<0.0001) afbraak (49.32%) en vermindering in
mutagenisiteit (42.47%) van die molekuul teweeggebring.
Beduidende (P<0.0001-0.05) afbraak van AFB1 is verkry na behandeling met
witvrot fungus kultuurekstrakte wat lakkase-ensiem bevat (17.10-76.00%), gesuiwerde
lakkase geproduseer deur Trametes versicolor (1 U/ml, 87.34%), sowel as rekombinante
lakkase geproduseer deur Aspergillus niger (118 U/L, 55.00%). Verder het die
behandeling van AFB1 met gesuiwerde lakkase-ensiem (1 U/ml) gelei tot verlies aan
mutagenisiteit van AFB1. Die afname in fluoressensie en mutageniese eienskappe van
die AFB1-molekuul na behandeling met die onderskeie mikrobiese preparate dui op
struktuurveranderings aan die furofuraan- en/of laktoonringe van die molekuul.
Hierdie studie lewer `n bydrae tot die ontwikkeling van geneties gemanipuleerde
mikrobiese rasse wat as `n belangrike biokontrole kan dien. Sulke rasse met
multifunksionele tegnologiese eienskappe, insluitend die afbraak van AFB1, kan die
kwaliteit, veiligheid en aanvaarbaarheid van voedsel verbeter.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/19429 |
| Date | 04 1900 |
| Creators | Alberts, Johanna Francina |
| Contributors | Van Zyl, W.H., Botha, A., Stellenbosch University. Faculty of Science. Dept. of Microbiology. |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | English |
| Type | Thesis |
| Format | 116 leaves : ill. |
| Rights | Stellenbosch University |
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