Thesis (MSc)--Stellenbosch University, 2001. / ENGLISH ABSTRACT: Lactic acid bacteria are present in many foods and beverages and are used as starter cultures in
the production of a variety of fermented products. Many of these bacteria produce ribosomally
synthesized antimicrobial peptides (bacteriocins), which inhibit the growth of bacteria genetically
closely related to the producer cell. Since many of these target bacteria include foodbome
pathogens such as Bacillus spp., Clostridium spp., Listeria spp., and Staphylococcus spp., the
practical importance of these peptides as food preservatives has been well documented and, in the
case of nisin and pediocin PA-I, commercially explored.
The increased demand from health conscious consumers for foods with no chemical
preservatives is putting renewed pressure on the producer to supply a "clean and green" product,
but with the same or even an extended shelf life. Various research groups are screening lactic
acid bacteria for production of novel broad-spectrum antimicrobial peptides or are exploring the
possibilities of altering known bacteriocins to inhibit Gram-negative bacteria, yeasts and molds.
Pediocin PD-I, produced by Pediococcus damnosus NCFB 1832, belongs to the class Ila
bacteriocins, i.e. heat-stable Listeria-active peptides, containing the YGNGV -consensus sequence
in the N-terminal region. Little is known about the production and mode of activity of pediocin
PD-I.
In this study, production of pediocin PD-I was significantly increased by optimizing the
growth medium, De Man Rogosa and Sharpe (MRS) broth. Addition of bacteriological peptone
(1.7%, w/v), manganous sulphate (0.014%, w/v) and Tween 80 (3%, v/v), and lowering of the pH
during fermentation stimulated pediocin PD-I production and the level of organic acids
produced. Maximum levels of bacteriocin activity were recorded at an initial pH of 6.7 in the
latter medium. Under these conditions the specific bacteriocin activity increased by a factor of
approximately six after 55 h of fermentation.
The effect of pediocin PD-I, plantaricin 423, produced by Lactobacillus plantarum 423, and
commercial grade nisin (Aplin and Barrett Ltd., Trowbrige, Wilts, England) was tested against
planktonic cells of Oenococcus oeni and a biofilm of the cells established on stainless steel
surfaces identical to those used in wineries. After 5 h of treatment with 3000 AU (arbitrary
units )/ml of each bacteriocin, all planktonic cells of 0. oeni in a modified Chardonnay must
medium were killed. All viable cells in the biofilm were killed after only 1 h in the presence of 3000 AU/ml of anyone of the bacteriocins. In addition, pediocin PD-I, plantaricin 423 and nisin
removed the biofilms from the surfaces and reduced the biomass either completely, as in the case
of pediocin PD-I, or by 58% and 50% as in the case of plantaricin 423 and nisin, respectively.
These same results were recorded after 5 h of treatment with 3000 AU/ml in a modified
Chardonnay must medium.
To our knowledge this is the first report of controlling biofilm formation of malolactic bacteria
on stainless steel surfaces with natural antimicrobial peptides. This implies that, apart from being
very effective in controlling the cell numbers of free-living cells of 0. oeni, the three
bacteriocins, especially pediocin PD-I, could also be used as natural sanitizers. The fact that the
production and activity levels ofpediocin PD-I could be increased without genetically modifying
the producer strain is an added advantage. / AFRIKAANSE OPSOMMING: Melksuurbakterieë is teenwoordig in verskeie soorte voedsel- en drankprodukte en word as
suurselkulture in die produksie van 'n verskeidenheid gefermenteerde produkte gebruik. Baie van
hierdie bakterieë produseer ribosomaal-vervaardigde antimikrobiese peptiede (bakteriosiene) wat
die groei van ander bakterieë, geneties naverwant aan die produserende organisme, inhibeer.
Omdat baie van hierdie bakterieë voedselpatogene soos Bacillus spp., Clostridium spp., Listeria
spp. en Staphylococcus spp. insluit, is die praktiese belang van hierdie peptiede reeds deeglik
ondersoek en word, soos in die geval van nisien en pediosien PA-I, kommersieel gebruik.
Die toenemende behoefte van die verbruiker na voedselprodukte met geen chemiese
preserveermiddels plaas nuwe druk op die vervaardiger om veilige voedselprodukte te produseer,
maar met dieselfde of selfs langer rakleeftyd. Verskeie navorsingsgroepe bestudeer
melksuurbakterieë vir die produksie van unieke antimikrobiese peptiede met 'n wye spektrum van
inhibisie en ondersoek ook die moontlikhede om hierdie bakteriosiene geneties te manipuleer ten
einde Gram-negatiewe bakterieë, giste en swamme te inhibeer.
Pediosien PD-l, geproduseer deur Pediococcus damnosus NCFB 1832, word as 'n klass na
bakteriosien geklassifiseer. Hierdie groep sluit in die hitte-stabiele Listeria-aktiewe peptiede, met
'n YGNGV-konsensus volgorde in die N-terminale deel van die peptied. Min is egter bekend oor
die meganisme van werking van hierdie bakteriosiene.
In hierdie studie is die produksie van pediosien PD-l betekenisvol verhoog met die
optimalisering van die vloeibare groeimedium De Man Rogosa en Sharpe (MRS). Die
toevoeging van bakteriologiese peptone (1.7%, miv), mangaan sulfaat (0.014%, miv) en Tween
80 (3.0%, v/v) en 'n afname in die pH gedurende groei het pediosien PD-l-poduksie gestimuleer
en sodoende ook die vlak van organiese sure wat geproduseer is. Maksimum vlakke van
bakteriosien-aktiwiteit is in hierdie medium met 'n aanvangs-pH van 6.7 waargeneem. Onder
hierdie omstandighede, en na 55 uur van fermentasie, het die spesifieke aktiwiteit van die
bakteriosien met 'n faktor van ongeveer ses verhoog.
Die effek van pediosien PD-l, plantarisien 423, geproduseer deur Lactobacillus plantarum
423, en 'n kommersiële graad nisien (Aplin and Barrett Ltd., Trowbride, Wilts, Engeland) is teen
die planktoniese selle van Oenococcus oeni en 'n biofilm van hierdie selle, gevestig op 'n vlekvrye
staaloppervlak identies aan wat in wynkelders gebruik word, getoets. Na 5 ure van behandeling met 3000 AB (arbitrêre eenhede)/ml van elke bakteriosien, is al die planktoniese selle van O. oeni
in 'n gemodifiseerde Chardonnay mos-medium vernietig. Alle lewensvatbare selle in die biofilm
is ook na slegs 1 uur in die teenwoordigheid van 3000 AE/ml van enige een van hierdie
bakteriosiene vernietig. Verdermeer het pediosien PD-I, plantarisien 423 en nisien ook die
biofilm op die vlekvrye staal-oppervlak verwyder. In die geval van pediosien PD-I is 'n totale
afname van die biomassa-oppervlak waargeneem, terwyl plantarisien 423 en nisien 58% en 50%
van die totale biomassa verwyder het. Hierdie resultate is na 5 ure van behandeling (3000
AE/ml) in 'n gemodifiseerde Chardonnay mos-medium waargeneem.
Sover ons kennis strek is hierdie die eerste verslag rakende die gebruik van natuurlike
antimikrobiese peptiede om biofilm-vorming deur appel-melksuurbakterieë op vlekvrye staal
oppervlaktes te beheer. Dit impliseer dat bakteriosiene, spesifiek pediosien PD-I, benewens die
beheer van planktoniese selle van appel-melksuurbakterieë, ook as natuurlike oppervlak-reinigers
gebruik kan word. Die feit dat die produksie en aktiwiteitsvlakke van pediosien PD-I verhoog
kon word sonder om die organisme geneties te modifiseer is 'n verdere voordeel.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/52272 |
| Date | 12 1900 |
| Creators | Nel, Hannes Augustinus |
| Contributors | Dicks, L. M. T., Wolfaardt, G. M., Stellenbosch University. Faculty of Science. Dept. of Microbiology. |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | Unknown |
| Type | Thesis |
| Format | 179 pages |
| Rights | Stellenbosch University |
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