Dissertation (PhD Agric)--University of Stellenbosch, 2004. / ENGLISH ABSTRACT: Malolactic fermentation (MLF) is conducted by lactic acid bacteria (LAB) and entails
the decarboxylation of L-malate to L-Iactate through a reaction catalysed by the
malolactic enzyme (MLE). The consequence of this conversion is a decrease in total
acidity. MLF plays a part in microbial stabilisation and due to the metabolic activity of
the bacteria the organoleptic profile of the wine is modified. In some wines MLF is
considered as spoilage, especially in warm viticultural regions with grapes containing
less malic acid. In addition to undesirable organoleptic changes, MLF can alter wine
colour, and biogenic amines may be produced. To induce MLF we provided
s. cerevisiae with the enzymatic activities required for MLF, which is then conducted
by the yeast during alcoholic fermentation. The malolactic enzyme-encoding gene
(mieD) was cloned from Pediococcus damnosus NCFB 1832, characterised and
expressed in S. cerevisiae. The activity of this enzyme was compared to two other
malolactic genes, mieS from Lactococcus lactis MG1363 and mleA from Oenococcus
oeni La11, expressed in the same yeast strain. All three recombinant strains of
S. cerevisiae converted L-malate to L-Iactate in synthetic grape must, reaching
L-malate concentrations of below 0.3 gIL within 3 days. However, a lower conversion
rate and a significant lower final L-Iactate level were observed with the yeast
expressing mieD. In order to inhibit MLF, we show that the growth of O. oeni, the
main organism responsible for MLF, could be safely repressed with a ribosomaly
synthesised antimicrobial peptide, pediocin PD-1, produced by P. damnosus NCFB
1832, without effecting yeast growth. Pediocin PD-1 is stable in wine at 4°C-100°C,
and ethanol or S02 does not affect its activity. The peptide was purified to
homogeneity and sequence analysis suggests that the peptide is a member of the
lantibiotic family of bacteriocins. The molecular mass was estimated by mass
spectroscopy to be 2866.7 ± 0.4 Da. Pediocin PD-1 forms pores in sensitive cells, as
indicated by the efflux of K+ from O. oeni, combined with inhibition of cell wall
biosynthesis, leading to cell lysis. Loss of cell K+was reduced at low temperatures,
presumably as a result of the increased ordering of the lipid hydrocarbon chains in
the cytoplasmic membrane. Although pediocin PD-1 is active over a broad pH range,
optimal activity was recorded at pH 5.0. The petide is, however, more stable
between pH 2.0 and 5.0, with the best stability observed between pH 3.0 and 4.0.
Pediocin PD-1 provides a safer biological alternative than chemical preservatives
such as S02. / AFRIKAANSE OPSOMMING: Appelmelksuurgisting (AMG) word deur sekere melksuurbakterieë (MSB) uitgevoer
en verwys na die dekarboksilering van L-malaat na L-Iaktaat, 'n reaksie gekataliseer
deur die appelmelksuurensiem (AME). AMG verlaag die suurvlakke in wyn, speel 'n
rol in mikrobiologiese stabiliteit, en verander die organoleptiese profiel van die wyn.
In sommige wyne word AMG beskou as bederf, veral in warm wynbou streke met
minder malaat in druiwe. AMG kan ongewenste organoleptiese veranderinge teweeg
bring, die wyn se kleur beinvloed, en tot die produksie van biogene amiene lei. Vir
die bevordering van AMG het ons S. eerevisiae met die ensiematiese aktiwiteit
benodig vir AMG voorsien wat dan veilig deur die gis tydens alkoholiese fermentasie
uitgevoer word. 'n AME-koderende geen (mIeD) is uit Pedioeoeeus damnosus NCFB
1832 gekloneer, gekarakteriseer en in S. Cerevisiae uitgedruk. Die aktiwiteit van die
ensiem is vervolgens vergelyk met die aktiwitet van twee ander AME gene, mIeS van
Laetoeoeeus laetis MG1363 en mleA van Oenoeoeeus oeni Lal1, uitgedruk in
dieselfde gisras. AI drie rekombinante gisrasse het L-malaat binne die bestek van
drie dae na L-Iaktaat omgeskakel en die finale L-malaat vlakke was minder as 0.3
gIL. Die tempo van omkakeling was egter laer in die gis wat die mIeD geen uitdruk en
die finale L-Iaktaat vlakke was veel laer. Om AMG te inhibeer is die groei van O.
oeni, die organisme hoofsaaklik verantwoordelik vir AMG, onderdruk deur die
byvoeging van 'n ribosomaal gesintetiseerde antimikrobiese peptied, pediocin PD-1,
geproduseer deur P. damnosus NCFB 1832. Gisgroei is nie geaffekteer nie.
Pediocin PD-1 is stabiel in wyn by temperature wat wissel tussen 4°C en 100°C, en
die aktiwiteit van die peptied word nie geaffekteer deur ethanol of S02 nie. Die
peptied is gesuiwer volgens In eenvoudige metode wat amoniumsulfaat-presipitasie
en katioon uitruilings-ehromatografie insluit. Aminosuur volgorde bepaling van
gesuiwerde peptied dui daarop dat pediocin PD-1 tot die lantibiotiese familie van
bakteriosiene behoort. Die molekulêre massa van die peptied, soos bepaal deur
massa spektroskopie, is 2866.7 ± 0.4 Da. Pediocin PD-1 vorm porieë in
selmembrane van sensitiewe selle soos aangedui deur die uitvloei van K+vanuit O.
oeni selle. Die peptied kombineer hierdie aksie met die inhibisie van selwand
biosintese wat lei tot sel lise. Verlies van sellulêre K+verminder by laer temperature,
waarskynlik as gevolg van verandering in die lipied- en protein inhoud van die
sitoplasmiese membraan. Alhoewel die peptied aktief is oor 'n breë pH grens, is die
antimikrobiese aksie optimaal by pH 5.0. Die peptied is meer stabiel tussen pH 2.0
en 5.0 en toon die beste stabiliteit tussen pH 3.0 en 4.0. Peiocin PD-1 is 'n veilige
biologiese alternatief vir chemiese preserveermiddels soos S02.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/50106 |
| Date | 12 1900 |
| Creators | Bauer, Rolene |
| Contributors | Dicks, L. M. T., Stellenbosch University. Faculty of AgriSciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology. |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | English |
| Type | Thesis |
| Format | 137 p. |
| Rights | Stellenbosch University |
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