Thesis (MSc) -- University of Stellenbosch, 2004. / ENGLISH ABSTRACT: Lactic acid bacteria play an essential role in the majority of fermented foods by producing
organoleptic compounds and increasing the shelf life. The best-studied antimicrobial compounds
are bacteriocins, i.e. ribosomally synthesized peptides. Most of these peptides have a narrow
spectrum of activity and are usually only active against bacteria from the same ecological niche.
The fact that all bacteriocins are degraded by proteolytic enzymes enlarges their potential use as
natural food preservatives. The ideal would be to replace or reduce chemical preservatives such
as sulfur dioxide, nitrates and nitrites.
Bacteriocins are classified into four groups according to their structural and functional
characteristics. Plantaricin 423, produced by Lactobacillus plantarum 423, is heat stable,
plasmid encoded, relatively small (3.5 kDa) and is classified as a class Iia bacteriocin. The
peptide is active from pH 1.0 to 10.0 and inhibits Gram-positive bacteria, including Lactobacillus
spp., Leuconostoc spp., Oenococcus oeni, Pediococcus spp., Enterococcus spp.,
Propionibacterium spp. and pathogens such as Bacillus cereus, Clostridium spp. and Listeria
monocytogenes.
Production of bacteriocins may occur constitutively or may be regulated by a cell-density
dependent system called quorum sensing. Plantaricin 423 is produced throughout logarithmic
growth, with no apparent change in production levels when the producer strain is cultured in the
presence of plantaricin 423 or Listeria innocua and Lactobacillus sakei. This led us to believe
that plantaricin 423 may be produced constitutively.
A reporter system was constructed which consisted of the plantaricin 423 promoter, P423,
fused to the luxAB genes and cloned into a shuttle vector, pTRKH2. The newly constructed
plasmid, pTAB4, was transformed to a bacteriocin-negative mutant of L. plantarum (423 B}
Despite several repeats, no luciferase activity was recorded and no RNA homologous to the
luxAB genes was detected.
The region necessary for expression of plantaricin 423 may be located stream-up of the -80
region homologous to the -80 and -40 conserved repeats of regulated class II bacteriocins.
Inclusion of the latter region in the reporter construct may result in the successful expression of
luxAB. / AFRIKAANSE OPSOMMING: Melksuurbakteriee speel 'n belangrike rol in die meeste gefermenteerde voedselsoorte deur
die produksie van organoleptiese komponente en die verlenging van rakleeftyd. Van aile
antimikrobiese komponente is bakteriosiene (ribosomaal gesintetiseerde peptiede) die beste
bestudeer. Hierdie peptiede het gewoonlik 'n nou spektrum van antimikrobiese werking en is
meestal aktief teen bakteriee in dieselfde ekologiese nis. Die feit dat bakteriosiene deur
proteolitiese ensieme in die spysverteringskanaal vernietig word, verhoog die potensiele gebruik
van bakteriosiene as preserveermiddels. Die ideaal sal wees om die konsentrasie van chemiese
preserveermiddels soos swaweldioksied, nitrate en nitriete te verlaag of rnoontlik te vervang met
bakteriosiene.
Bakteriosiene word in vier groepe op grond van hul strukturele en funksionele
karaktereienskappe geklassifiseer. Plantarisien 423, geproduseer deur Lactobacillus plantarum
423, is hitte-stabiel, word deur 'n plasmied gekodeer, is relatief klein (3.5 kDa) en sorteer onder
die klas Iia bakteriosiene. Die peptied is aktief oor 'n wye pH-reeks (pH 1.0-10.0) en inhibeer
Gram-positiewe bakteriee, insluitend Lactobacillus spp., Leuconostoc spp., Oenococcus oeni,
Pediococcus spp., Enterococcus spp., Propionibacterium spp. en patogene soos Bacillus
cereus, Clostridium spp. en Listeria monocytogenes.
Produksie van bakteriosiene kan konstitutief plaasvind of kan gereguleer word deur 'n seldigtheids-
afhanklike sisteem naamlik "quorum sensing". Plantarisien 423 word regdeur
logaritmiese groei geproduseer, met geen verandering in produksievlakke wanneer die
produserende stam in die teenwoordigheid van plantarisien 423 of Listeria innocua en
Lactobacillus sakei gekweek word nie. Dit het gelei tot die hipotese dat plantarisien 423
moontlik konstitutief geproduseer word.
'n Verklikkersisteem bestaande uit 'n fusie van die plantarisien 423 promoter, P423, aan die
luxAB gene is gekonstrueer en in die pendelplasmied pTRKH2 gekloneer. Die nuutgekonstrueerde
plasmied, pTAB4, is na 'n bakteriosien-negatiewe mutant van L. plantarum
(stam 423 B-) getransfonneer. Ten spyte van etlike herhalings kon geen lusiferase-aktiwiteit
opgespoor word nie en kon ook geen homologie in die RNA met die luxAB gene opgespoor
word nie.
Dit is moontlik dat die area nodig vir uitdrukking van plantarisien 423 verder stroom-op van
die -80 area, homoloog aan die -80 en -40 gekonserveerde herhalings van reguleerbare klas II
bakteriosiene, gesetel is. Insluiting van laasgenoemde area in die verklikker-konstruk mag lei tot
die suksesvolle uitdrukking van luxAB.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/50111 |
| Date | 12 1900 |
| Creators | Cohen, Francisca |
| Contributors | Dicks, L. M. T., Wolfaardt, G. M., Albertyn, J., 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 | 89 p. : ill. |
| Rights | Stellenbosch University |
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