Thesis (PhD)--University of Stellenbosch, 2000. / ENGLISH ABSTRACT: Worldwide, bacteriocins, particularly those produced by food-related lactic acid bacteria, are
receiving attention due to the possible use of these peptides as natural preservatives in food,
replacing potentially harmful chemical preservatives.
Bacteriocins are ribosomally synthesized proteins or peptides that inhibit closely related
microorganisms. Most bacteriocins produced by lactic acid bacteria are small, heat resistant
peptides that inhibit other Gram-positive bacteria, including food-borne pathogens such as
Listeria monocytogenes, Bacillus cereus, Clostridium perfringens and Staphylococcus aureus,
but do not inhibit Gram-negative bacteria, molds or fungi. Bacteriocins are produced as
inactive prepeptides that become active after the N-terminal leader peptide is cleaved off.
Small heat resistant bacteriocins are either lantibiotics (Class I), containing unusual posttranslationally
modified amino acids, or peptides that are non-Ianthionines (Class II). The
Class II bacteriocins are further divided into four different groups: Class lIa, the anti-listerial
bacteriocins containing the YGNGV consensus sequence in the N-terminal of the protein,
Class lib, bacteriocins consisting of two peptides, Class IIc, bacteriocins that are secreted via
the sec pathway, and Class lid, bacteriocins that do not belong in the previous three
subgroups.
A bacteriocin producing lactic acid bacterium was isolated in our laboratory from
traditionally home fermented South African sorghum beer. The producing bacterium was
found to be a facultative heterofermentative Lactobacillus sp. and was identified as
Lactobacillus plantarum or Lactobacillus pentosus by using the API 50 CHL carbohydrate
fermentation system and numerical analysis of total soluble cell protein patterns. RAPD-PCR
analysis identified the strain as L. plantarum, but 16S rRNA sequencing confirmed its
identification as L. pentosus.
The bacteriocin, first designated plantaricin 423 and later bacteriocin 423, was identified as
a Class lIa small heat resistant anti-listerial bacteriocin containing the YGNGV consensus
motif. Bacteriocin 423 inhibited a variety of Gram-positive bacteria, including Lactobacillus
spp., Leuconostoc spp., Oenococcus oeni, Pediococcus spp., Enterococcus spp.,
Propionibacterium spp., Staphylococcus spp., Bacillus spp., Clostridium spp. and Listeria spp.
The bacteriocin was inactivated by proteolytic enzymes and active over a wide pH range (pH
1-10). Bacteriocin 423 lost 50 % of its activity after autoclaving for 15 min at 121°C, but was
not affected by lesser heat treatments.
Bacteriocin production was increased by optimizing the growth medium, which consisted of
glucose, tryptone, yeast extract, potassium phosphate, sodium acetate, ammonium citrate, manganese sulphate, Tween 80 and casamino acids.
The bacteriocin was found to be plasmid-encoded. Genetic analysis of the bacteriocin
operon indicated a high percentage of homology to the operon of another Class lIa
bacteriocin, pediocin PA-1, although the structural genes of the two bacteriocins were
markedly different. The structural gene of bacteriocin 423 was amplified by PCR and cloned
into a yeastJE. coli vector between the ADH1 promoter and terminator sequences and fused
in-frame to the MFa1 secretion signal sequence. Saccharomyces cerevisiae transformed with
this plasmid expressed the bacteriocin.
The sequence of prebacteriocin 423 (MMKKIEKL TEKEMANIIGGKYYGNGVTCGKHSCSVN
WGOAFSCSVSHLANFGHGKC) is similar, but not identical to any other reported Class lIa
anti-listeria I peptide. / AFRIKAANSE OPSOMMING: Bakteriosiene, veral dié wat deur melksuurbakterieë geproduseer word, wek belangstelling
as gevolg van die moontlike gebruik van hierdie natuurlike antimikrobiese proteiëne as
preserveermiddels in voedselprodukte, in plaas van potensieël gevaarlike chemiese
preserveermiddels.
Bakteriosiene is ribosomaal-vervaardigde proteiëne wat naverwante bakterieë inhibeer.
Die meeste bakteriosiene wat deur melksuurbakterieë geproduseer word, is klein en
hittebestand. Hierdie bakteriosiene inhibeer ander Gram-positiewe bakterieë, insluitend
patogene soos Listeria monocytogenes, Bacillus cereus, Clostridium perfringens en
Staphylococcus aureus, maar inhibeer nie Gram-negatiewe bakterieë, giste of swamme nie.
Bakteriosiene word as onaktiewe prepeptiede geproduseer, wat ge-aktiveer word wanneer die
N-terminale leierpeptied afgesplits word. Klein hittebestande bakteriosiene is óf lantibiotika
(Klas I), met ongewone aminosure, óf normale peptiede (Klas II). Laasgenoemde klas kan
verder in vier groepe verdeel word. Klas lIa is anti-listeriese bakteriosiene met fn YGNGVaminosuurvolgorde
in die N-terminale kant van die peptied. Klas lib sluit in bakteriosiene wat
uit twee peptiede bestaan. Klas lie is sec-afhanklike bakteriosiene, en Klas lid sluit in al die
bakteriosiene wat nie in die eerste drie groepe geklassifiseer kan word nie.
'n Bakteriosien-produserende melksuurbakterie is uit tradisionele tuisgefermenteerde Suid-
Afrikaanse sorghumbier geïsoleer. Die bakterie is as 'n fakultatief heterofermentatiewe
Lactobacillus sp. geïdentifiseer. Die bakterie is verder as 'n Lactobacillus plantarum of
Lactobacillus pentosus geïdentifiseer deur middel van die API 50 CHL-koolhidraat
fermentasiesisteem en numeriese analiese van totale oplosbare selproteiënprofiele. Met
RAPD-PCR analiese is die organisme as L. plantarum geïdentifiseer, maar 168 rRNA
nukleotiedopeenvolging het die identiteit van die organisme as L. pentosus bevestig.
Bakteriosien 423, aanvanklik geklassifiseer as plantaricin 423, is fn klein Klas lIa,
hittebestande en anti-listeriese bakteriosien met die YGNGV motief, wat verskeie Grampositiewe
bakterieë inhibeer. Bakteriosien 423 het verskeie Gram-positiewe organismes
geïnhibeer, onder andere Lactobacillus spp., Leuconostoc spp., Oenococcus oeni,
Pediococcus spp., Enterococcus spp., Propionibacterium spp., Staphylococcus spp., Bacillus
spp., Clostridium spp., en Listeria spp. Proteolitiese ensieme inaktiveer die bakteriosien. Die
peptied was oor 'n pH reeks van 1-10 aktief. Outoklavering vir 15 min by 121°C het die
aktiwiteit van die peptied halveer, maar die bakteriosien is nie geïnaktiveer met ander
hittebehandelings nie.
Produksie van die bakteriosien is verhoog deur die groeimedium te optimiseer. Die groeimedium het bestaan uit glukose, triptoon, gisekstrak, kaliumfosfaat, natriumasetaat,
ammoniumsitraat, mangaansulfaat, Tween 80 en casaminosure.
Die bakteriosien se genetiese determinante is op In plasmied gesetel. Genetiese analiese
van die bakteriosien operon het 'n hoë homologie met In ander Klas lIa bakteriosien, pediocin
PA-1, getoon, maar die strukturele gene van die twee bakteriosiene verskil merkbaar. Die
strukturele geen van bakteriosien 423 is met PKR ge-amplifiseer en in 'n gistE. coli-vektor
tussen die ADH1 promotor- en termineerderopeenvolgings, in leesraam met die MFa1
sekresiesein, gekloneer. Saccharomyces cerevisiae wat met hierdie plasmied getransformeer
is, het bakteriosien 423 uitgedruk. Die aminosuurvolgorde van prebakteriosien 423
(MMKKIEKL TEKEMANIIGGKYYGNGVTCGKHSCSVNWGOAFSCSVSHLANFGHGKC) is verwant
aan, maar nie identies aan, ander Klas lIa anti-listeriese peptiede.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/51652 |
| Date | 12 1900 |
| Creators | Van Reenen, Carol A. (Carol Ann) |
| Contributors | Dicks, L. M. T., Van Zyl, W. H., 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 | 178 p. : ill. |
| Rights | Stellenbosch University |
Page generated in 0.0029 seconds